To control Ca2+ signaling, these organelles are often observed close to influx and release sites and may be tethered near Ca2+ transporters. In this study, we used high-resolution, wide-field fluorescence imaging to investigate the regulation of Ca2+ signaling by mitochondria in large numbers of endothelial cells (∼50 per field) in intact arteries from rats. We observed that mitochondria were mostly spherical or short-rod structures and were distributed widely throughout the cytoplasm. The density of these organelles did not increase near contact sites with smooth muscle cells. However, local inositol trisphosphate (IP3)-mediated Ca2+ signaling predominated near these contact sites and required polarized mitochondria. Of note, mitochondrial control of Ca2+ signals occurred even when mitochondria were far from Ca2+ release sites. Indeed, the endothelial mitochondria were mobile and moved throughout the cytoplasm. Mitochondrial control of Ca2+ signaling was mediated by ATP production, which, when reduced by mitochondrial depolarization or ATP synthase inhibition, eliminated local IP3-mediated Ca2+ release events. ROS buffering did not significantly alter local Ca2+ release events. These results highlight the importance of mitochondrial ATP production in providing long-range control of endothelial signaling via IP3-evoked local Ca2+ release in intact endothelium.Endothelial cells are reported to be glycolytic and to minimally rely on mitochondria for ATP generation. Rather than providing energy, mitochondria in endothelial cells may act as signaling organelles that control cytosolic Ca Endothelial cells (ECs)1–versus oxidative phosphorylation may enable ECs to meet rapid changes in energy demands. As endothelial cells are required to grow into hypoxic surroundings during angiogenesis, a reliance on anaerobic metabolism may enable ECs to form new vessels. These observations have led to the proposal that endothelial mitochondria act primarily as essential signaling organelles rather than being energy providers , adhesion molecules (e.g. von Willebrand factor), and clotting factors, occur in a Ca2+-dependent manner. In various other cell types, Ca2+ signals are regulated by mitochondria. Uptake of the ion by mitochondria may promote Ca2+ release from IP3R  may cluster   Ca2+ release and mitochondrial Ca2+ uptake in RBL-2H3 cells. Conversely, disrupting the linkage by limited proteolysis decreases mitochondrial Ca2+ uptake  , demonst2+ release events arising from the ER may occur preferentially at sites of contact between endothelial cells and smooth muscle cells ) , rapid (∼0.25 s) events arising from IP3Rs and referred to as pulsars ) . Ca2+ si pulsars . MEPs thsynthase , IP3Rs . Localitivation , 46 and tivation , raising tissues .2+ release events in endothelial cells in intact blood vessels obtained from rats. We found that local endothelial Ca2+ signals preferentially initiate at contact sites with smooth muscle cells, and the Ca2+ signals share the pharmacological profile of Ca2+ pulsars. However, the local signals have a substantially different temporal profile from pulsars. The occurrence of local Ca2+ signals at MEPs requires polarized mitochondria. Inhibition of mitochondrial respiration eliminates Ca2+ activity at MEPs. Surprisingly, neither mitochondrial positioning nor density correlates with sites that give rise to cytosolic Ca2+ events, as might be predicted if tethering were required for mitochondrial control of Ca2+ release. Ca2+ event initiation sites are, on average, 0.9 μm from the nearest mitochondrion and on occasion up to 5 μm away. These results indicate that close coupling is not required for mitochondrial control of Ca2+ release in the endothelium and that control is exerted over distance. Inhibition of the ATP synthase eliminated local Ca2+ signaling events, whereas buffering reactive oxygen species had little effect. Thus, mitochondria exert long-range control of IP3-mediated intracellular Ca2+ signaling dynamics in native endothelial cells via ATP production.To address this issue, we examined spontaneous Ca2+ signaling was assessed in endothelial cells of intact second-order mesenteric arteries (∼150-μm diameter). Time-series image recordings (20 Hz) of the endothelium were obtained using a high-NA (1.3) ×100 microscope objective and a large-format EMCCD camera . In opened arteries (en face preparations), this experimental set-up provided a field of view of ∼17,720 μm2 with a pixel size of 130 × 130 nm projected onto the endothelium. On average, 52 ± 2 whole or partial endothelial cells were visualized in each field of view . Thus, we calculated the density of endothelial cells to be on the order of 2000 cells/mm2, in agreement with our previous estimates (Castimates .A and Movie S1) and baseline-corrected  fluorescence recordings revealed extensive Ca2+ activity in mesenteric artery endothelial cells. The signals formed a continuum of events that ranged from small, highly localized focal increases in Ca2+ (akin to Ca2+ puffs) to traveling spatial gradients (waves) that progressed completely or partly  through cells. Partial Ca2+ waves were the predominant form of Ca2+ activity. On average, 12.5 ± 1.5% of ECs exhibited spontaneous Ca2+ activity . Often, a single location would give rise to repetitive Ca2+ events, although the properties  of events that arose from single sites varied (C–E).In the absence of stimulation with pharmacological agents or mechanical forces, close visual inspection of raw  the initiation sites from which Ca2+ activity originated and applied an automated Ca2+ signal analysis algorithm. The algorithm was adapted from our previous work  For each initiation site, we extracted baseline-corrected Ca2+ signals  and then automatically identified Ca2+ events . The algorithm identifies Ca2+ events using peaks in the time derivative of the F/F0 signal. Ca2+ events were taken as changes in the time derivative of the F/F0 signal that exceeded a threshold value of 10 times the S.D. value of the baseline signal fluctuation. This threshold (10-fold baseline noise) was chosen empirically, as being large enough to exclude noise fluctuations but small enough to faithfully detect low-amplitude Ca2+ signals. A suitable baseline period was automatically identified for each trace . Following event extraction, we then used a least-squares optimization algorithm to fit an exponentially modified Gaussian function  to each Ca2+ peak to measure event parameters. Histograms  show that each measured parameter , rise time, fall time, and spatial spread) may be described by a continuous log normal distribution. The mean values of each were as follows: frequency ; amplitude ; FDHM ; rise time ; fall time , and spatial spread .To analyze spontaneous local endothelial Caous work  . Removal of external Ca2+ had no significant effect on either the density of Ca2+ event initiation sites  or the density of Ca2+ events , suggesting that the events arose via Ca2+ release from the internal store rather than Ca2+ influx . In support, the SERCA inhibitor, cyclopiazonic acid , abolished basal endothelial Ca2+ events . CPA reduced the initiation site density from 59 ± 7 to 5 ± 10 sites min−1 mm−2, whereas the event density was reduced from 115 ± 15 to 5 ± 2 events min−1 mm−2 . Ca2+ events that persisted after CPA incubation could still be described by an exponentially modified Gaussian function , inset). The broad-spectrum TRPV channel antagonist, ruthenium red (RuR) , was without effect on either the density of Ca2+ event initiation sites  or the density of Ca2+ events . We have previously shown that this concentration of RuR (5 μm) is sufficient to inhibit endothelial Ca2+ activity induced by the specific TRPV4 agonist, GSK1016790A  , also failed to inhibit basal endothelial Ca2+ activity .Spontaneous Ca1016790A . Similar2+ release from the endoplasmic reticulum may occur primarily via IP3Rs in the vascular endothelium . 2-APB reduced the Ca2+ event initiation site density from 36 ± 6 to 2 ± 0 sites min−1 mm−2 and the Ca2+ event density from 76 ± 21 to 3 ± 0 events min−1 mm−2 . Although 2-APB blocks IP3Rs in native endothelial cells, it may also inhibit Ca2+ entry pathways . Caffeine reduced the Ca2+ event initiation site density from 50 ± 11 to 4 ± 2 sites min−1 mm−2, and the Ca2+ event density from 86 ± 20 to 5 ± 2 events min−1 mm−2 . As a control, we also show that caffeine inhibited Ca2+ release induced by photolysis of caged IP3 .Caothelium . To invepathways . Therefoe 10 mm; , B and C2+ events did not arise from mechanisms involving voltage-activated Ca2+ channels located on smooth muscle cells. In support, spontaneous Ca2+ events were present in isolated endothelial patches (A). Furthermore, the Ca2+ channel blocker, nimodipine (10 μm), did not reduce the density of initiation sites  or indeed of events themselves  in intact arteries . This concentration of nimodipine (10 μm) is sufficient to fully block voltage-dependent Ca2+ currents in smooth muscle cells (–m K+) contraction of isolated arteries  occur extensively in unstimulated endothelium  and confined (spread of ∼15 μm2) events when compared with the Ca2+ signals observed in the present study   where MEPs occur.In murine mesenteric arteries, transient nonpropagating IPothelium . Pulsarsnt study . The diftensity) . Notwithdark regions in the fluorescence field . To highlight the location of these IEL holes, we smoothed, inverted, and colorized autofluorescence images so that IEL holes were shown as blue on a black background . IEL holes were distributed extensively across the IEL, with 819 ± 58 holes/mm2 corresponding to a fenestrated area of 7.0 ± 0.6% of the elastic lamina. This measurement is similar to the estimate in murine mesenteric arteries . A pooled analysis of all IEL holes observed in 27 fields of endothelial cells (n = 9) demonstrated the lognormal distribution of IEL hole area .The IEL of intact small mesenteric arteries was visualized using autofluorescent emission from the elastin layer upon UV illumination. In autofluorescence images, holes in the IEL appear as ce field A, left. ckground A, right.arteries  but subsarteries . The aveA shows a typical image obtained when Ca2+ event initiation sites from a field of endothelial cells (identified in F/F0 recordings) are overlaid on an image of the underlying IEL holes. In all 27 fields in which endothelial Ca2+ and the underlying IEL were investigated, Ca2+ events occurred close to IEL holes . Measuring the distance from each event initiation site to the nearest IEL hole revealed that, on average, ∼50% (n = 225 distinct event sites) of Ca2+ events initiated directly at an IEL hole location , and ∼85% initiated with 5 μm of an IEL hole.Fig. 72+ signals. We thus next investigated whether or not local Ca2+ events occurred more often at holes in the IEL than would be predicted from a random overlap of the two. To this end, two separate tests were performed to determine whether Ca2+ events were statistically more likely to occur close to an IEL hole than elsewhere in the cell.However, fenestration of the IEL is extensive, and the apparent co-localization may be expected from a random distribution of IEL holes and local CaB; p < 0.05, two-sample Kolgorov–Smirnov test). This result suggests that Ca2+ events are more likely to occur close to an IEL than elsewhere in the cell.First, we pooled all data to generate (and then compare) cumulative probability distributions for the distances between initiation sites and IEL holes and the distances between all locations and IEL holes. In this analysis, we found that the separation between IEL holes and initiation sites was significantly less than the separation between IEL holes and all other sites . This resulted in 1000 sets of random Ca2+ event locations, and for each 1000 permutations, the centroid–centroid distances were once again measured. The average minimum distance between an IEL hole and a randomly redistributed event initiation site  was significantly higher than that measured from the real data . Thus, spontaneous Ca2+ events occurred more often near IEL holes than would be expected if the Ca2+ events initiated randomly throughout the cytoplasm.Next, we performed Monte Carlo simulations (permutation tests) to generate random distributions of Ca00 times , C–E. Th2+ events described are IP3-mediated Ca2+ pulsars, albeit with a different kinetic profile from those occurring in murine mesenteric artery. These events occur at initiation sites that are closer to MEPs (IEL holes) than expected from a random distribution. However, the large mean distance between MEPs and initiation sites (2.4 μm) suggests that direct coupling of initiation sites to MEPs is unlikely. It may be that in rat mesenteric arteries, Ca2+ events manifest as local propagating waves to couple activity at the initiation site to effector proteins that are located within MEPs.Taken together, the results presented thus far suggest that the Ca2+ signaling in a variety of excitable and nonexcitable cell types  (240 nm) to visualize the organelles . This value is similar to that observed in native smooth muscle cells , we observed extensive movement of the majority of endothelial mitochondria (Movie S3). In recordings of shorter duration (5 min), mitochondrial motion was less pronounced but was observed nonetheless in two of six recordings (A–C). The period for Ca2+ imaging was 1 min. The findings suggest that mitochondria in native endothelial cells are largely mobile structures and so are unlikely to be tethered to Ca2+ release sites.As a first step in examining the relationship between the location of mitochondria and Cacordings , A–C. Th2+ release event initiation sites. Because of the possibility of mitochondrial movement, we dual loaded the endothelium with the Ca2+ indicator, Cal-520/AM (5 μm), and TMRE (240 nm) to enable us to record both Ca2+ activity and mitochondrial position from the same field of endothelial cells . However, the separation between IEL holes (where Ca2+ events occur) and mitochondria was not significantly different from the separation measured between IEL holes and all other sites . This result suggests that mitochondria are not coupled to Ca2+ release sites. Notably, a substantial percentage of Ca2+ release sites (9%) had no mitochondria located within a 2.5-μm radius, and ∼2% of Ca2+ release sites had no mitochondria within a 5-μm radius . The averaged separation between local Ca2+ signal initiation sites and nearest mitochondria was slightly lower  than expected from a random distribution of event initiation sites . However, there was no correlation between sites of Ca2+ event initiation and mitochondrial density . The large mean separation (0.9 μm) between mitochondria and Ca2+ event initiation sites strengthens the view that direct modulation of Ca2+ initiation site activity by mitochondria is unlikely.During short imaging sessions (1-min duration), we observed that mitochondria appeared to be positioned close to many Caon sites , A and B2+ signaling, we investigated the effects of the mitochondrial uncoupler, CCCP (5 μm), or the complex 1 inhibitor, rotenone (2 μm). CCCP reduced the initiation site density from 35 ± 8 to 3 ± 2 sites min−1 mm−2, whereas the event density was reduced from 48 ± 7 to 3 ± 2 events min−1 mm−2 . Rotenone reduced the Ca2+ event initiation site density from 39 ± 7 to 3 ± 3 sites min−1 mm−2 and the Ca2+ event density from 60 ± 17 to 17 ± 6 events min−1 mm−2 . When the proton gradient across the mitochondrial membrane is impaired by mitochondrial inhibitors, such as CCCP or rotenone, the ATP synthase may reverse and consume ATP. Therefore, in the next series of experiments, the effects of the mitochondrial ATP synthase inhibitor, oligomycin (6 μm), on the CCCP-induced decreases in basal endothelial Ca2+ activity were examined. Oligomycin by itself was without effect on the mitochondrial membrane potential, as assessed by TMRE fluorescence . In contrast, when CCCP was subsequently added, there was a rapid loss of punctate mitochondrial staining and a significant decrease in TMRE fluorescence . Rotenone also significantly depolarized mitochondria, as revealed by a reduction in TMRE fluorescence , consistent with its role as a mitochondrial complex 1 inhibitor . Nor did oligomycin prevent the reduction in Ca2+ activity seen when rotenone was applied by itself (B).When applied in combination with CCCP, oligomycin did not prevent the reduction in Cay itself A. Nor diy itself B.2+ activity caused by these mitochondrial toxins arose from depletion of intracellular Ca2+ stores, we examined the Ca2+ response to the ionophore, ionomycin, in a Ca2+-free physiological saline solution (PSS) . The ionomycin releasable store content was unaffected by rotenone (with oligomycin present) or oligomycin applied alone. However, the ionomycin-evoked Ca2+ increase was significantly reduced by CCCP (with oligomycin present). It is possible that CCCP may inhibit Ca2+ release from the store, as has been shown previously for other nonexcitable cells  (i.e. without simultaneously uncoupling mitochondria using CCCP or rotenone). Remarkably, we found that inhibition of the ATP synthase nearly eliminated basal Ca2+ dynamics (A); oligomycin reduced the density of initiation sites from 44 ± 6 to 1 ± 0 sites min−1 mm−2 and the density of events from 93 ± 23 to 1 ± 0 events min−1 mm−2 . Oligomycin also inhibited spontaneous Ca2+ activity in isolated endothelial cell patches .Taken together, these results suggest that polarized mitochondria are required for IPactivity . Howeverdynamics A; oligom patches B; n = 3.2+ event initiation sites  or the density of Ca2+ events , suggesting that ATP derived from glycolysis is not required to maintain basal endothelial Ca2+ dynamics . These results suggest that ATP produced by mitochondria, and not glycolysis, facilitates IP3-mediated Ca2+ signaling in native endothelial cells.Removal of external glucose had no significant effect on either the density of Ca3-mediated Ca2+ signaling in endothelial cells. To this end, we investigated the effects of the nontargeted, cell-permeable reactive oxygen species (ROS) scavenger, TEMPOL (100 μm), and the mitochondria-targeted ROS scavenger, mitoTEMPO (50 μm). We found that nontargeted ROS scavenging was ineffective in reducing the density of Ca2+ event initiation sites  or of Ca2+ events  in native endothelial cells (B). Targeting the mitochondrial site of oxidative stress resulted in a modest decrease (∼20%) in both the Ca2+ event initiation site density  and the Ca2+ event density  in native endothelial cells (C).In a final series of experiments, we investigated whether oxidative stress induces basal IPal cells B. Targetal cells C.3-mediated Ca2+ signals, in the endothelium of intact small arteries, initiate at sites that are close to MEPs and are controlled by polarized mitochondria. When the mitochondrial membrane potential is depolarized, spontaneous Ca2+ signals cease. However, in contrast to many other cell types, mitochondrial control of local signals does not require the organelles to be positioned close to the Ca2+ release site. Rather, mitochondria exert long-range control of Ca2+ signaling in the vascular endothelium. The most likely candidate for the control is mitochondrial ATP production , which is generated by proton pumps . Together, the membrane potential and the proton gradient form the transmembrane potential of hydrogen ions used to make ATP. Collapse of the mitochondrial membrane potential inhibits ATP production, prevents mitochondrial Ca2+ uptake, and alters the production of ROS. In the present study in native endothelial cells, the uncoupler, CCCP, or the complex I inhibitor, rotenone, each collapsed the mitochondrial membrane potential and inhibited IP3-mediated basal Ca2+ signaling. Each drug (CCCP or rotenone) was effective in inhibiting Ca2+ signals when applied alone or in combination with oligomycin to prevent reversal of the ATP synthase. The ATP synthase blocker oligomycin, applied alone (as expected), did not decrease ΔΨm and yet also inhibited local Ca2+ signals. These results suggest that ATP production may maintain local endothelial Ca2+ signals in intact arteries. IP3-induced Ca2+ release may be regulated by a variety of cofactors and processes, which include ATP  and limit a Ca2+-dependent feedback process that controls Ca2+ release . The close apposition exposes the organelles to a high local Ca2+ concentration as the ion is released from the store, which overcomes the low affinity of the uniporter for Ca2+ . The stpes –103–, 61, 62.2+ events. Previous studies have also demonstrated co-localization of Ca2+ signals and MEPs  Act 1986, United Kingdom), under UK Home Office regulations. All experiments used second- or third-order mesenteric arteries obtained from male Sprague–Dawley rats , euthanized by overdose of COm NaCl, 4.7 mm KCl, 2.0 mm MOPS, 1.2 mm NaH2PO4, 5.0 mm glucose, 2.0 mm pyruvate, 0.02 mm EDTA, 1.17 mm MgCl2, 2.0 mm CaCl2, adjusted to pH 7.4 with NaOH. Small mesenteric arteries were then cleaned of connective tissue and fat, removed from the mesenteric bed, cut open using microscissors, and pinned endothelial side-up on a Sylgard block. The endothelium was then preferentially loaded with the fluorescent Ca2+ indicator, Cal-520/AM (5 μm with 0.04% Pluronic F127 and 0.26% DMSO in PSS) at 37 °C for 30 min. Following incubation, preparations were gently washed in PSS and mounted in a custom chamber designed for use on an inverted microscope  and spatial resolution  using an inverted fluorescence microscope  equipped with a ×100 objective  and a large-format (1024 × 1024 13-μm pixels) EMCCD camera . Cal-520/AM was excited with 488-nm wide-field epifluorescence illumination provided by a monochromator . The resulting image field (∼133 × 133 μm) enabled us to visualize connected networks of ∼50 whole or partial endothelial cells. Following equilibration (30 min), Ca2+ activity was recorded at room temperature for periods of 60 s. In experiments investigating the effects of pharmacological intervention, Ca2+ recordings were obtained from at least three separate fields of view per animal. Each of these regions was imaged both before and after the intervention. In these experiments, drugs were added directly to the imaging chamber, incubated for 20 min (unless otherwise indicated), and remained present throughout recordings. In experiments using a glucose-free PSS, glucose was substituted with d-mannitol on an equimolar basis. In experiments using a Ca2+-free PSS, Ca2+ was substituted with Mg2+ on an equimolar basis, and 1 mm EGTA was included.Immediately following euthanasia, the mesenteric bed was removed and placed in PSS composed of 145 mcroscope , 114. En2+ indicator, Cal-520/AM, and loaded at 37 °C for 30 min. The digestion protocol was designed to leave intact sheets of endothelial cells, which could be identified by their morphology (A) and by their response to ACh (B).Cut open arteries were cut into three or four smaller segments, and the segments were transferred into a PSS solution containing collagenase (2 mg/ml). The segments were incubated in this solution for 20 min at 37 °C and then gently washed three times in PSS. Following the wash steps, endothelial sheets were dispersed by triturating with a fire-polished glass pipette. The suspension was then transferred to an imaging chamber, and isolated endothelial sheets were allowed to attach to the glass coverslip for 1 h. After this period of time, the solution was exchanged for one containing the Carphology A and by e to ACh B.2+ signals recordings were analyzed using a custom semiautomated Python-based analysis adapted from our previous work  preprocessing of Ca2+ imaging data; 2) identification of sites of Ca2+ activity; 3) extraction of Ca2+ signals from active sites; and 4) analysis of Ca2+ event parameters. Each step is described below.Local Caous work . The pro2+-imaging recordings were preprocessed in FIJI as described previously . The preprocessing resulted in an image stack where positive pixel values indicate an increase in fluorescence above the average. Ca2+ event initiation sites were then identified manually. To do this, ΔF/Favg image stacks were then scrolled through, and each Ca2+ event was marked by a circular ROI that was centered over the point of initiation. Events often occurred repeatedly at a single site. In these cases, only a single ROI was positioned over the active site. Each ROI was added to the FIJI ROI manager, and, after ensuring that all Ca2+ activity was marked, the center coordinates of the ROIs were saved for subsequent Ca2+ signal extraction, as described below.Caeviously . First, 2+ signals were extracted from the raw fluorescence intensity (F) image stacks, using 30-pixel (∼4-μm) diameter circular ROIs positioned at the initiation site of each Ca2+ event as described above. The signals were extracted and processed using a modification of our previously published algorithm for batch processing of two-dimensional Ca2+ data , third-order polynomial Savitzky–Golay filter, corrected for baseline drift using asymmetric least squares fitting,F) traces were expressed as fractional changes from baseline (F/F0) by dividing the fluorescence intensity trace by the average value of a 100-frame (5-s) baseline period (F0). The baseline period was automatically determined for each trace as the portion of signal exhibiting the lowest S.D. This was achieved by applying a rolling S.D. (100 frames) and a rolling summation (100 frames) to each trace. The minimum of the rolling summation corresponds to the center of the “quietest” portion of the F/F0 trace. Ca2+ events were then automatically identified (A) using a zero-crossing detector on the derivative F/F0 traces (2+ events from noise. A positive peak in the derivative function corresponds to the positive edge of a Ca2+ event. A negative peak (nadir) in the derivative function corresponds to the negative edge of a Ca2+ event. The zero-crossings associated with a peak and a preceding nadir  in the derivative trace indicate, respectively, the start, peak, and end of an event in the corresponding Ca2+ trace. The zero-crossing times were used to extract those parts of the original Ca2+ trace that contained Ca2+ events. Event parameters  were then extracted by fitting each detected Ca2+ event with a Gaussian function.Temporal Caa2+ data . For eacentified A using a0 traces . A thres2+ events and myoendothelial gap junctions, images of the internal elastic lamina underlying endothelial cells and Ca2+ imaging data were recorded. The IEL was visualized using autofluorescence from 390-nm excitation light and single images generated by averaging 100-frame recordings obtained at 10 Hz. In rat mesenteric arteries, “holes” in the IEL correlate with the presence of myoendothelial gap junctions and may present a low-resistance pathway for diffusible factors  initiation site to IEL hole separation was calculated.To determine whether the extent of co-localization of Caanalysis . First, m) (m) in all perfusion solutions. Images of TMRE fluorescence (excited at 555 nm) were acquired at 20 Hz using the TE2000U microscope system described above. In experiments assessing mitochondrial morphology, TMRE fluorescence was recorded (1-min periods) from at least three separate fields of view per animal. For each field of view, single images were generated by averaging 100 frames, and mitochondrial morphology was determined using MicroP , the same single field of endothelial cells was imaged throughout, and TMRE fluorescence was measured after performing a 10-pixel-wide background subtraction using FIJI. In experiments examining mitochondrial motility, mitochondria were imaged for periods of 5 or 30 min at 2 Hz. Mitochondrial movement tracks were generated using TrackMate  . TMRE wa nm . TMRrackMate .2+ event initiation sites, mitochondrial TMRE fluorescence and Ca2+ activity were each recorded in the same field of endothelial cells. Single images of mitochondria were generated by averaging 100 frames (5 s) of the full recording. To facilitate comparison of localization, mitochondria were contrast-enhanced by converting images to a binary form. This was achieved by applying (in sequence) an unsharp mask filter (2-pixel radius), a rolling ball (5-pixel diameter) background subtraction, a mean filter (1-pixel radius), a linear contrast enhancement, an adaptive local contrast enhancement, a median filter (1-pixel radius), and finally an intensity threshold (Otsu's automatic method). The extent of co-localization between mitochondria and the Ca2+ event initiation site was then determined as described above for IEL holes.To assess the extent of spatial coupling between mitochondria and Ca2+ signaling was examined in response to photolysis of caged IP3. The endothelium of en face arteries was dual-loaded with Cal-520/AM (5 μm), and a membrane-permeant caged IP3, caged IP3 4,5-dimethoxy-2-nitrobenzyl (10 μm), 0.02% Pluronic F-127, and 0.35% DMSO in PSS for 30 min at 37 °C. Endothelial Ca2+ imaging was then performed at 10 Hz, using an inverted fluorescence microscope  equipped with a ×40 objective  and a large-format (1024 × 1024 13-μm pixels) EMCCD camera  with a 325-nm projected pixel size at focal plane. Cal-520/AM was excited with 488-nm wide-field epifluorescence illumination provided by an LED illumination system . The EMCCD was configured to record from the central 512 × 512 pixels, resulting in a field of view of ∼166 × 166 μm . Photolysis of caged IP3 was achieved using a xenon flashlamp  attached directly to the TE300 microscope . To create probability distributions, data were pooled from all experimental animals within each treatment group. In general, summary data are presented graphically as individual data points  and in the text as the grand mean with the S.E. indicated. Non-Gaussian data (identified using the D'Agostino–Pearson omnibus test) were log normal. Log normal data were transformed (log10), and mean values for each experimental unit were calculated on the logarithmic scale and then back-transformed to their original scale for presentation. In the text, log normal data are presented back-transformed grand means with 95% confidence intervals provided for completeness.Except for probability distributions, the 2+ response to ionomycin or photolysis of caged IP3 or experiments utilizing isolated sheets of endothelial cells, Ca2+ imaging data were collected from at least three different fields of endothelial cells from three different arteries per rat. In Ca2+ experiments in which ionomycin or caged IP3 was used, a single field of endothelial cells was studied per animal. A single field of endothelial cells was also studied  in experiments examining the effects of CCCP/rotenone on the mitochondrial membrane potential. Data were analyzed using Student's t test or repeated measures analysis of variance, with Dunnett's multiple-comparison test, as indicated throughout. All statistical analysis was performed using GraphPad Prism version 6 . A p value of <0.05 was considered statistically significant. Data supporting the findings of this study are available from the corresponding authors on request.With the exception of experiments assessing the CaC. W. and J. G. M. developed the concept. C. W., M. D. L., H. R. H., X. Z., and C. B. performed the experiments. C. W. and C. D. S. wrote the analysis software. C. W., C. S., C. B., and J. G. M. analyzed the data. C. W. and J. G. M. drafted the manuscript. C. W., C. D. S., J. M. G., and J. G. M. revised and edited the manuscript. C. W., C. D. S., J. M. G., and J. G. M. sourced funding. All authors approved the final version of the manuscript.


Schistocerca gregaria (S. gregaria), pheromones are considered to be crucial for governing important behaviors and processes, including phase transition, reproduction, aggregation and swarm formation. The receptors mediating pheromone detection in olfactory sensory neurons (OSNs) on the antenna of S. gregaria are unknown. Since pheromone receptors in other insects belong to the odorant receptor (OR) family and are typically co-expressed with the “sensory neuron membrane protein 1” (SNMP1), in our search for putative pheromone receptors of S. gregaria, we have screened the OR repertoire for receptor types that are expressed in SNMP1-positive OSNs. Based on phylogenetic analyses, we categorized the 119 ORs of S. gregaria into three groups (I–III) and analyzed a substantial number of ORs for co-expression with SNMP1 by two-color fluorescence in situ hybridization. We have identified 33 ORs that were co-expressed with SNMP1. In fact, the majority of ORs from group I and II were found to be expressed in SNMP1-positive OSNs, but only very few receptors from group III, which comprises approximately 60% of all ORs from S. gregaria, were co-expressed with SNMP1. These findings indicate that numerous ORs from group I and II could be important for pheromone communication. Collectively, we have identified a broad range of candidate pheromone receptors in S. gregaria that are not randomly distributed throughout the OR family but rather segregate into phylogenetically distinct receptor clades.In the desert locust  Antheraea polyphemus as a major protein component in membranes of pheromone-responsive OSNs  for 15 min at 4 °C. Subsequently, the sections were hybridized in 100 μL hybridization solution 1  supplemented with the relevant digoxigenin- and biotin-labeled riboprobes. Alternatively, sections were pre-hybridized for 1 h at 60 °C in hybridization buffer 2 . A volume of 100 μL hybridization buffer 2 supplemented with the relevant RNA probes was evenly applied onto each slide with adhered tissue sections. Next, a coverslip was placed on top and slides were incubated at 60 °C overnight (18–20 h) in a box equipped with paper towels soaked with 50% formamide. On the following day, slides were washed twice for 30 min in 0.1× SSC at 60 °C; then each slide was treated with 1 mL 1% blocking reagent (Roche Diagnostics) in TBS  for 40 min at room temperature in a box to keep the sections moist. Visualization of hybridized digoxigenin-labeled riboprobes was performed by using an alkaline phosphatase-conjugated antibody against digoxigenin in combination with HNPP/Fast Red (Roche Diagnostics). To visualize probes labeled with biotin, streptavidin conjugated to horseradish peroxidase was utilized together with tyramides coupled to fluorescein . The slides were analyzed with a Zeiss LSM510 Meta laser scanning microscope  and the acquired confocal image stacks were further processed by ZEN 2009 software (Carl Zeiss Microscopy). The images presented in Figures 2–5 and in FISH experiments were carried out as described previously ,29,30. B+ RNA was extracted with the Dynabeads mRNA purification kit following the manufacturer’s protocol. Finally, poly(A)+ RNA samples were eluted with 22 µl H2O and RNA concentrations were measured by means of an Epoch microplate spectrophotometer . Synthesis of first strand cDNA was carried out with 250 ng of poly(A)+ RNA, 2 µL 50 µM oligo(dT)20 primer (Thermo Fisher Scientific), 2 µL 10 mM 2’-deoxynucleoside 5’-triphosphate (dNTP) solution mix  and RNase-free H2O. Following a 5-min incubation at 65 °C, 8 µL 5× SSIV Buffer (Thermo Fisher Scientific), 2 μL 100 mM 1,4-dithiothreitol , 2 µL RNaseOut (Thermo Fisher Scientific) and 1 µL Superscript IV Reverse Transcriptase (Thermo Fisher Scientific) were added. Synthesis of cDNA was conducted at 52 °C for 50 min followed by 10 min at 80 °C. In parallel, control samples were prepared for which no reverse transcriptase was added. To investigate a potentially sex-dependent expression of the group II SgreOR types co-expressed with SNMP1, the entire antennae from 10 male and 10 female, sexually mature individuals were removed surgically. After freezing the antennae immediately in liquid nitrogen, RNA was extracted utilizing Trizol reagent as recommended by the supplier. Next, poly(A)2O, 5 µL 10× Titanium Taq PCR Buffer , 0.5 µL of each primer (100 µM), 1 µL 10 mM dNTP solution mix, 2 µL first strand cDNA and 0.5 µL 50× Titanium Taq DNA Polymerase (Takara Bio). Thermocycling was conducted using the following parameters: 1 min at 97 °C succeeded by 35 cycles with 97 °C for 30 s and 3 min at 68 °C. After the last cycle, samples were incubated at 68 °C for 3 min. The resulting PCR products were visualized using agarose gels supplemented with ethidium bromide. PCR amplicons of the predicted molecular size were extracted using the Monarch DNA Gel Extraction Kit (New England Biolabs) and sent to GATC Biotech  for sequencing.For the subsequent amplification of sequences encoding actin (GenBank accession number: AEV89776) or the OR types SgreOR84-86, SgreOR88, SgreOR90-97, SgreOR101-103, SgreOR109-114, SgreOR116 and SgreOR118 in PCR experiments, the oligonucleotide primers given in S. gregaria [Locusta migratoria (LmigOrs) [In this study, we extended our recent efforts to identify candidate pheromone receptors of gregaria  and examgregaria  utilizingregaria , 138 ORsLmigOrs)  and seveLmigOrs) . AccordiL. migratoria [L. migratoria [L. migratoria  were found to be not co-expressed with SNMP1. The images of the corresponding FISH experiments for some of these ORs are exemplarily depicted in L. migratoria. From 21 SgreORs of this category, 18 were analyzed by two-color FISH. The results of these approaches are shown for some of these ORs in At first, ORs of the paralogous clusters/pairs  were analyzed. In our previous study , it was S. gregaria, group III comprises by far the largest number (70) of receptors. In order to explore a potential co-expression with SNMP1 in antennal OSNs, we have previously [Within the OR repertoire of eviously  analyzedeviously . These feviously  studies,A compilation of the results acquired by the FISH experiments, including the findings of our previous study , is presS. gregaria. The results for several SgreORs from group II are depicted in For lepidopteran species, it has previously been shown that receptors for certain pheromones, especially for sex pheromones, are often expressed in a sex-specific or at least sex-biased manner ,9. To apS. gregaria by screening the large repertoire of SgreORs for co-expression with SNMP1 that is considered a marker for pheromone-responsive OSNs [In this study, attempts were made to identify candidate pheromone receptors from the desert locust ive OSNs ,5. Togetive OSNs , 83 SgreD. melanogaster, out of the 37 OR types that are expressed in the antenna [D. melanogaster, the desert locust has a comparatively large number of ORs that are co-expressed with SNMP1. This finding raises the question why the number of ORs co-expressed with SNMP1 is that high in S. gregaria. In this regard, it is important to note that locusts are characterized by a phase polyphenism and are well known for their ability to form giant swarms [S. gregaria may further substantiate the concept that in locusts, a large variety of behaviors and physiological processes rely on intraspecific communication via a broad spectrum of chemical signals. However, some of the SgreOR types co-expressed with SNMP1 could be involved in the reception of non-pheromonal compounds. In particular, since a few ORs from D. melanogaster, namely Or19a or Or83c, that are co-expressed with SNMP1 [So far, the repertoire of ORs that are co-expressed with SNMP1 has been sparsely analyzed in other insects. In the dipteran species  antenna , only ni antenna . Consequt swarms ,35. For t swarms ,35. Moret swarms ,35,36,37th SNMP1 ,34 are ath SNMP1 ,39. TherL. migratoria. The relatively high sequence identity observed between receptor types of this group from different locusts is reminiscent of pheromone receptors from several lepidopteran species that are supposed to mainly utilize orthologous ORs for pheromone detection [L. migratoria and non-paralogous SgreORs that lack such an orthologue , a marker for pheromone-responsive OSNs. In total, a relatively high number of 33 SgreORs were found to be co-expressed with SNMP1 in OSNs, suggesting a large repertoire of pheromone receptors in S. gregaria. Strikingly, almost all of the SNMP1-co-expressed OR types belong to the phylogenetically classified groups I and II of SgreORs. Moreover, most members of these two groups were observed to be co-expressed with SNMP1. By contrast, analyzing numerous SgreORs from group III, which comprises almost 60% of all SgreORs, only three receptor types were expressed in SNMP1-positive OSNs. Thus, receptors co-expressed with SNMP1 in S. gregaria are not evenly distributed throughout the entire OR repertoire, but cluster in distinct phylogenetic OR groups. In summary, our results indicate that in contrast to SgreORs from group III, a larger number of OR types from groups I and II could be involved in pheromone communication of S. gregaria.In search for candidate pheromone receptors, we have analyzed about two-thirds of the


ADP; from 33.0 ± 2.4 to 18.4 ± 3.8 and 32.8 ± 1.3 to 5.9 ± 2.7 pmol/s/mg wet weight; −44.2 ± 11.4% vs. −82.0 ± 8.1%, in young and aged mice, respectively). Baseline CRC was lower in old animals and IR similarly decreased the CRC in both groups . Further, IR-induced ROS production tended to be higher in aged mice. In conclusion, aging exacerbated the deleterious effects of IR on skeletal muscle mitochondrial respiration, potentially in relation to an increased oxidative stress.Cycles of ischemia-reperfusion (IR) that occur during peripheral arterial disease (PAD) are associated with significant morbi-mortality, and aging is an irreversible risk factor of PAD. However, the effects of advanced age on IR-induced skeletal muscle mitochondrial dysfunction are not well known. Young and aged mice were therefore submitted to hindlimb IR (2 h ischemia followed by 2 h reperfusion). Skeletal muscle mitochondrial respiration, calcium retention capacity (CRC) and reactive oxygen species (ROS) production were determined using high resolution respirometry, spectrofluorometry and electronic paramagnetic resonance. IR-induced impairment in mitochondrial respiration was enhanced in old animals (V Ischemia-reperfusion (IR) cycles that occur during peripheral arterial disease (PAD) induce skeletal muscle injury resulting in lifestyle-limiting disability, including exercise intolerance and in the most severe cases, limb amputation. PAD is also often associated with other cardiovascular diseases including myocardial infarction, stroke and cognitive dysfunctions. With more than 200 million people diagnosed in the world, PAD is a public health issue. PAD prevalence is high in aged people and although individuals over the age of 70 present the highest risk, revascularization in nonagenarian patients appeared effective as long as the patient was not fully dependent ,4,5,6. HPAD pathophysiology clearly demonstrated a major role for skeletal muscle mitochondrial dysfunction and oxidative stress, which promote cell damage and death. Thus, oxidative phosphorylation capacity is reduced, hampering adenosine triphosphate (ATP) generation ,11,12,13The aim of this study was therefore to investigate whether old age enhances IR-induced skeletal muscle mitochondrial alterations, analyzing both mitochondrial oxidative and calcium retention capacities, together with reactive oxygen species (ROS) production in young and aged mice submitted to unilateral hindlimb IR.Experiments were performed on young (23 ± 1 weeks old) and aged (84 ± 1 weeks old) male C57Bl6J mice. The mice were housed in a neutral temperature environment (22 ± 2 °C) on a 12 h light-dark cycle. Standard food and water were available ad libitum. All experiments were performed in agreement with the French laws for animal use and care and the guidelines from directive 2010/63/EU. The experiments were approved by the local ethics committee and the French Research Minister (agreement number 2018041811246867).®, Esternay, France) to maintain animal body temperature near 37 °C. Spontaneous ventilation was allowed through the oxygen delivering mask. Depth of anesthesia was checked by tail clamp before more noxious stimuli, and controlled by adapting isoflurane concentrations.Mice were anesthetized in a hermetic cage, ventilated with gas mixture of 4% isoflurane  and oxygen. They were placed on heating blankets  and A-Sham (n = 5)), were submitted to 4 h anesthesia. Two other young and aged groups were submitted to 2 h ischemia with a tourniquet placed around the right hindlimb, at the level of the groin, followed by 2 h reperfusion (Y-IR (n = 7), A-IR (n = 7), respectively). Ischemia was ascertained by visual observation of cyanosis of the limb and mice were sacrificed at the end of the experiment [Mice were divided into four groups . Young aperiment .2 2.5 mM, MgSO4 1.2 mM, NaHCO3 25 mM, KH2PO4 1.04 mM, D(+) glucose 5.6 mM, Na-HEPES 20 mM, pH 7.4 at 4 °C) allowing preservation of muscle fibers in a physiological environment at 4 °C. Samples were either gently dissected on ice under a dissecting microscope for mitochondrial respiration and CRC measurements or cut into 1–2 mm3 slices for assessment of ROS production.The superficial gastrocnemius muscles , characterized by a greater susceptibility to IR-induced damage , were ha2EGTA 2.77 mM, K2EGTA 7.23 mM, Na2ATP 6.04 mM, MgCl2 6.56 mM, taurine 20 mM, Na2Phosphocreatine 12.3 mM, imidazole 20 mM, dithiothreitol 0.5 mM, K-methane sulfonate 50 mM, pH 7.0 at 4 °C) with saponin (50 µg/mL). Then fibers were rinsed with agitation for 10 min at 4 °C in the buffer S. This technique allowed mitochondrial morphology to be kept and the functional cellular environment to be preserved [After dissection, muscle fibers were permeabilized by incubation under stirring for 30 min at 4 °C in a buffer S . Then fibers were placed in 2 mL of buffer R+ in the oxygraphic chamber and basal oxygen consumption (V0) was measured. Subsequently, several substrates were added in order to specifically activate different complexes of the mitochondrial electron transport chain (ETC). Addition of a saturating amount of adenosine diphosphate (ADP) (2 mM) allowed measuring complex I, III and IV activity (VADP). Then, a saturating amount of succinate (25 mM) allowed studying complex I, II, III and IV activity (VSucc). Finally, addition of N,N,N′,N′-tetramethyl-p-phenylenediamine  and ascorbate (0.5 mM) specifically activated complex IV (VTMPD/Asc). The ratio VADP/V0 was calculated to evaluate the degree of coupling between oxidation and phosphorylation. Results were expressed as pmol/s/mg wet weight.Measuring oxygen consumption in permeabilized fibers was performed using a Clark electrode in a thermostated oxygraphic chamber at 37 °C with continuous stirring . Briefly, fibers (3–4 mg wet weight) were incubated twice for 5 min with agitation at 4 °C in a buffer R+  containing ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (5 μM) and bovine serum albumin (2 mg/mL).Opening of the mPTP was assessed by monitoring CRC of skeletal muscle mitochondria under energized conditions, as previously described . BrieflyPermeabilized ghost fibers were incubated at 24 °C in a quartz tank with continuous stirring in 1 mL of CRC buffer containing calcium green-5N fluorescent probe . The reaction was started by the addition of a calcium pulse (20 mM), following by calcium pulses every 5 min until it was necessary. After each pulse, a peak of extramitochondrial calcium was recorded and a rapid uptake by the mitochondria was observed, resulting in a decrease in extramitochondrial calcium concentration to near basal level. When mitochondria reached the maximal calcium loading threshold, opening of mPTP happens and mitochondrial calcium is released, resulting in an abrupt increase in extramitochondrial calcium concentration. The amount of calcium necessary to trigger mPTP opening was calculated from a standard curve relating calcium concentrations to the fluorescence of calcium green-5N. At the end of the experiment, muscle fibers were gathered, dehydrated at 150 °C for 15 min, and weighted. Results were expressed as µmol/mg dry weight.2·) and other ROS of a spin probe 1-hydroxy-3-methoxycarbonyl-2, 2, 5, 5-tetramethyl-pyrrolidine . Muscle fragments were incubated for 30 min in Krebs-HEPES buffer containing deferoxamine (25 μM), diethyldithiocarbamate (5 μM) and CMH (200 µM) in a thermoregulated incubator at 37 °C under gas mix  and controlled pressure . Then the reaction was stopped on ice and all experiment measures of oxidized probe concentration were performed at 15 °C in disposable capillary tubes from 40 µL of supernatant, using the e-scan spectrometer , as described previously. Detection of ROS was conducted under the following settings, center field 3461.144 g, microwave power 21.85 mW, modulation amplitude 2.40 g, sweep time 5.24 s (10 scans), sweep width 60 g, number of lag curve points 1. The signal amplitude is calculated and the concentration of CM· is determined from standard calibration curve of CM. At the end of the experiment, muscle fragments were gathered, dehydrated at 150 °C for 15 min, and weighted. Results were expressed in μmol/min/mg dry weight.The electron paramagnetic resonance spectroscopy is one of the best techniques used to detect the “instantaneous” presence of free radical species in the samples, by oxidation from superoxide anion . Such impairment was enhanced in old mice V0  and VADP   A,B.Succ decreased from 43.7 ± 3.0 to 35.0 ± 2.8 and from 40.1 ± 1.5 to 30.2 ± 5.2 pmol/s/mg wet weight, in sham and IR young and aged mice, respectively. A main effect of IR (p = 0.0198) was observed, without a specific effect of aging (p = 0.2683)  C.TMPD/Asc) was activated , representing the degree of coupling between oxidation and phosphorylation, remained unchanged in all groups . IR further and similarly reduced the CRC in both groups .IR did not significantly modify ROS production . However, ROS tended to increase in aged animals  difference in mitochondrial respiration and ROS production between young (6 months old) and aged (21 months old) sham mice. These data were similar to others studying mitochondrial respiration ,48 and oIn young mice, as expected, although ROS production was not significantly increased, as has been observed previously in a similar setting reporting no ROS production at all ,21, we oConsidering aging, few studies specifically investigated a possible enhanced sensitivity of old muscle as compared to young muscles in response to lower limb IR and therefore analyzed concomitantly of both young and aged muscles in such a setting ,20,21,22Interestingly, relatively little is known on the effect of aging on mPTP opening susceptibility after IR in skeletal muscle. We observed that unlike mitochondrial respiration, the IR-induced CRC decrease was not enhanced in old mice. IR-related CRC alterations were nevertheless significant but, they were similar in young and old mice. Whether this might be related to the already decreased baseline CRC value in old muscle or if it is species specific, deserves further studies. Interestingly, even if heart and skeletal muscles differ on some aspects, according to our data, isolated cardiac mitochondria of aged rats showed increased mPTP opening susceptibility after IR . This miSeveral limitations deserve to be acknowledged. We did not investigate gender-associated differences that might have modulated both aging and IR-effects on skeletal muscles. This deserves to be studied. Further, although such a preclinical model is well-characterized and exhibits pathophysiological features similar to those observed in patients, this is an experimental study performed in healthy mice without key cardiovascular risk factors such as diabetes, hypertension etc. Thus, although the message of an enhanced susceptibility to IR-damage in aging might be even stronger when taking into account other risk factors, caution should be applied when extrapolating these data to humans.The present study provides evidence that aging exacerbates the deleterious effects of IR in skeletal muscle, further impairing mitochondrial respiration. Although not affecting the mitochondrial CRC, already impaired in old mice, such enhanced susceptibility to IR of aged muscles should likely be taken into account when treating older patients suffering from PAD.


Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected  Various protocols have been published for the extraction of DNA from phlebotomines, including methods that eliminate DNA-associated proteins by using detergents and salts ,2,3, or Lutzomyia spp. sand flies by PCR amplification [Lutzomyia cacophony gene [L. longipalpis sand flies. The protocol, which we here refer to as pAC, uses detergent (SDS), proteinase K and phenol-chloroform extraction .4.CRITICAL STEP: Add 450 µL buffer TESCa + pK , vortex for 1 min, and incubate at 50 °C for 8 h, vortexing for 1 min every 30 min.5.CRITICAL STEP: Add 500 µL chloroform:isoamyl alcohol (C:IAA) (24:1 v/v) and mix vigorously by inversion for 2 min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~480 µL) to a new 1.5 mL microcentrifuge tube.6.CRITICAL STEP: Add 500 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2 min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~460 µL) to a new 1.5 mL microcentrifuge tube.7.CRITICAL STEP: Add 700 µL C:IAA (24:1 v/v) and mix vigorously by inversion for 2 min. Immediately centrifuge at 14,000 rpm for 5 min. Transfer the supernatant (~400 µL) to a new 1.5 mL microcentrifuge tube.8.Add 0.1 volumes (~40 µL) 3 M Sodium Acetate (NaOAc) pH 5.2 and 2.5 volumes (~1 mL) 100% ethanol, and gently mix by inversion for 1 min.PAUSE STEP and OPTIONAL STEP: We found that after adding NaOAc and ethanol, results improved when the sample was immediately centrifuged . Nevertheless, due to the length of the previous stages (~9 h), the protocol can be paused here and the sample stored overnight (ON) at −20 °C.9.Centrifuge at 14,000 rpm for 20 min and discard the supernatant by inversion.10.Add 500 µL 70% ethanol and centrifuge at 14,000 rpm for 5 min. Discard the supernatant by inversion and dry the pellet at 50 °C for 5 min. Resuspend the pellet in 10 µL double-distilled water.2(CH2OH)3, 121.14 g/mol) TRIS buffer  12H25NaO4S, 288.38 g/mol) Sodium Dodecyl Sulfate  EDTA  Calcium chloride dihydrate 2O)Double-distilled water  Chloroform 3COONa, 82.03 g/mol) Sodium acetate  Glacial acetic acid  Absolute ethanol Vortex Water bath High-speed bench-top centrifuge Micropipettes p1000, p200, p20 2.Composition: 30 mM Tris-HCl pH 8; 10 mM EDTA; 1% SDS; 5 mM CaCl2, autoclave, and then add the SDS.Calculate the necessary volumes for each stock solution. Add and mix the Tris-HCl pH 8, EDTA, and CaClBelow we give an example :Lutzomyia constitutive cacophony gene (~225 bp) [L. longipalpis adults from Posadas (Argentina) using the pAC protocol; the negative control was ddH2O. The variations we assayed and the effects they produced are mentioned below . As previously mentioned, we found that internal control PCR results were variable for individual sand flies processed with the pAC protocol (results not shown). For this reason, we assayed various modifications to optimize DNA extractions from one sand fly . The qua~225 bp) ,13 , here referred to as buffer TESCa  in general yielded better results ; Table 1ntration . Furthere enzyme . In addie enzyme . This sae enzyme . 2O. Due to the length of the first three stages (~9 h), the protocol was paused in the fourth step . Amplification was variable for the samples processed with buffer TES .To evaluate these modifications, we processed specimens with the different lysis buffers   and founHaving determined that buffer TESCa and the previous modifications , consistently improved DNA extractions, we also analyzed if we could reduce the incubation periods with proteinase K in this new lysis buffer, and eliminate the ON incubation at −20 °C. As we had found previously, longer incubation periods with pK improved PCR amplification, and incubation at −20 °C seemed to have little effect ; Table 1(1)an 8-h incubation with proteinase K in buffer TESCa at 50 °C;(2)vigorous mixing by inversion during the extraction with solvents step; and(3)2O and a 1:5 dilution was used for the PCR reactions. The complete and detailed optimized protocol is described in “precipitation with alcohol with no ON incubation at −20 °C . PelletsTo summarize, the main modifications for the final optimized DNA extraction protocol consisted of: DNA obtained using our method is suitable for long-term conservation, since individual sand fly DNA extracts were stored at −20 °C and used as a template as much as 6 years later in PCR reactions which yielded positive results.Lutzomyia spp. from Brazil and Argentina, and the internal control amplifications were successful  optimized DNA extraction from individual sand flies when compared to the original pAC protocol, and enabled us to consistently obtain positive amplification results with the internal control primers. Moreover, we used this optimized protocol to extract DNA from individual field-captured ccessful . 2+ to the lysis buffer that improves DNA extraction. Our results also suggest that pK activity is reduced when the lysis buffer contains EDTA but no calcium , in acco


Scientific Reports 10.1038/s41598-018-31873-w, published online 13 September 2018Correction to: This Article contains errors in Tables 2, 5 and 6.b’ should read ‘Krakenbd’In Table 2, the subheading ‘KrakenIn Table 5, for the MetaPhlAn2 (Unix) method, the sensitivity and PPV values should read as 78% and 54% respectively. For the MIDAS (Unix) method, the sensitivity and PPV values should read as 78% and 21% respectively.a has been incorrectly included in the subheading ‘CLC Genomics Workbencha’, which should read ‘CLC Genomics Workbench’In Table 6, the subscript note


Extracellular vesicles are nanosized vesicles that are under intense investigation for their role in intercellular communication. Extracellular vesicles have begun to be examined for their role in disease protection and their role as disease biomarkers and/or vaccine agents. The goal of this study was to investigate the effects of alcohol exposure on the biogenesis and composition of extracellular vesicles derived from the cervical cancer line, HeLa. The HeLa cells were cultured in exosome-free media and were either mock-treated (control) or treated with 50 mM or 100 mM of alcohol for 24 h and 48 h. Our results demonstrated that alcohol significantly impacts HeLa cell viability and exosome biogenesis/composition. Importantly, our studies demonstrate the critical role of alcohol on HeLa cells, as well as HeLa-derived extracellular vesicle biogenesis and composition. Specifically, these results indicate that alcohol alters extracellular vesicles’ packaging of heat shock proteins and apoptotic proteins. Extracellular vesicles serve as communicators for HeLa cells, as well as biomarkers for the initiation and progression of disease. The World Health Organization (WHO)-International Agency for Research on Cancer (IARC) 2009) has classified alcoholic beverages as a Group 1 Carcinogen (carcinogenic to humans). A study completed by Madsen et al. found that in the absence of alcohol consumption, the prevalence of vaginal squamous cell carcinomas (VV-SCC) greatly decreased 009 has c,4,5 and/Recent studies demonstrated that body fluids contain substantial amounts of EVs with sizes varying between 30 and 1000 nm. Three main types of EVs have been described. First, nanosized exosomes (30–150 nm), which are derivatives of the endosomal system; second, microvesicles (100–1000 nm), which are produced from outward budding of the plasma membrane ; third, Discovered in the early 1980s, nanosized exosomes are heterogeneously-shaped vesicles that are released from the cell’s plasma membrane via exocytosis into extracellular environments ,25. ExosExosomes are released by most cell types and mediate targeting in normal cells under physiological and pathophysiological conditions, including different types of cancer ,33,34. E2 under standard humidified conditions.Human cervical epithelial (HeLa) cells were purchased from American Type Culture Collection (ATCC)  and cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) with 10% fetal bovine serum (FBS), 10 mM L-glutamine, and 1% penicillin-streptomycin . Exosome-free DMEM/F12 media was made in the same manner as DMEM/F12 using exosome-free FBS from System Biosciences . The cell line was incubated at 37 °C and 5% CO5 cells/per dish with 6 mL of DMEM/F12) . The following day, media was removed and replaced with exosome-free DMEM/F12. The dishes were dosed with DMEM/F12 exosome-free media only (control treatment) and DMEM/F12 exosome-free media containing 200-proof 50 mM and 100 mM EtOH . The media was collected at 24 h or 48 h.HeLa cells were washed with 5 mL of sterile 1× phosphate-buffered saline (PBS) , removed with 0.05% trypsin) , and plated at 5 × 105 cells/per dish with 6 mL of DMEM/F12. The following day, media was removed, and 6 mL exosome-free DMEM/F12 were added to the dishes. Cells were treated with 0.05% trypsin, scraped, and collected at 24 h or 48 h. Cells were stained with 0.4% trypan blue solution, and viability was measured using the trypan blue dye in Cell Countess . Percent viability = [1.00 − ] × 100.HeLa cells were grown to 70–80% confluency and seeded into tissue culture dishes at 5 × 10g at 4 °C for 10 min using a Sorvall© RT 6000 refrigerated centrifuge . The media was collected, and the cell pellet was discarded. The media was spun again at 2600× g at 4 °C for 10 min using a Sorvall© RT 6000 refrigerated centrifuge. The media was then filtered through a 10-mL syringe with a 25-mm syringe filter, with a porosity of 0.22 µm. Six milliliters of PBS was added to the media and centrifuged at 20,000× g for 45 min in a SW41T1 swinging bucket rotor at 4 °C using a Beckman Coulter Optima ™ L-70K Ultracentrifuge . To collect the EVs, the media was collected and centrifuged for 110,000× g for 70 min in a SW41T1 swinging bucket rotor at 4 °C using a Beckman Coulter Optima ™ L-70K Ultracentrifuge. The supernatant was removed, and approximately 500 µL of resuspended EVs were recovered from the sample. Isolated EVs were quantitated using Lowry quantitation methods [EVs were isolated from the media following EtOH exposure at different concentrations. EVs were also isolated from cells not treated and used as controls. Four to five milliliters of conditioned medium was collected for exosome purification. In brief, extracellular vesicles were isolated as previously described . After cThe size of HeLa-derived EVs was determined by Nanosight tracking analysis (NTA), by measuring Brownian motion to particle size distribution and count, using the Nanosight NS300 Sub-micron Particle Imaging System . Ten milliliters of 1× PBS diluted (1:1000) samples were injected into the machine sample chamber, and EVs were measured at room temperature (RT). The analysis of data was completed and recorded by the NTA software. The means ± SEM were recorded and analyzed for each given reading frame of the five independent experiments.To analyze EV-associated proteins, EVs were mixed with 5× sample loading buffer, boiled, and resolved on a 4–12% Bis-Tris sodium dodecyl sulfate gel, followed by transfer and blocking on a polyvinylidene difluoride membrane . Blotting was performed with clathrin and Hsp70 primary antibodies . Incubation with secondary antibody was performed using horseradish peroxidase (HRP)-conjugated goat anti-mouse 1:2,000 dilution . Proteins were detected using an enhanced chemiluminescence kit  and the signals were developed on a Bio-Rad ChemiDoc™ XRS+ System .Cell lysates were evaluated via dot blot analysis. Zero-point-eight micrograms of cell lysate were lysed with lysis buffer , boiled, and bound to nitrocellulose membranes for 10 min. Samples were blocked in Pierce Fast-Blocker  with 0.09% Tween-20 for 5 min. After blocking, primary antibodies Rab 5  and Rab 7  were added to the samples for incubation. Samples were incubated for 1 h at RT. Nitrocellulose blots were washed three times with 0.09% Tween-20 in 1× PBS for 10 min. Goat anti-rabbit (H+L) secondary antibody HRP  was added in blocking solution  with 0.09% Tween-20 in 1× PBS) for 1 h of shaking at RT. The blots were washed three times with 0.09% Tween-20 in 1× PBS for 10 min. The nitrocellulose membranes were developed using Super Signal West Femto Maximum Sensitivity Substrate . The signals were developed on a Bio-Rad ChemiDoc™ XRS+ System .o-phenylenediamine dihydrochloride (OPD) peroxidase substrate  and measured at OD 405 nm using an automated ELISA plate reader.In order to investigate EV-associated proteins, ELISAs were performed. ELISA plates were coated with EVs collected from 24 h or 48 h with concentrations of 0 mM, 50 mM, and 100 mM or control (blocking buffer). Forty milligrams of samples were bound overnight at 4 °C on a 96-well plate with bicarbonate buffer (pH 9.5). EVs were blocked for 1 h in 0.05% BSA + Tween-20 (blocking buffer) at 4 °C. Incubated plates were then washed three times, and the detection antibody was prepared. One hundred microliters of primary antibody  with blocking buffer were then added and incubated at RT for 2 h. ELISA plates were then washed three times, and secondary antibody, HRP-conjugated goat anti-mouse  or HRP-conjugated goat anti-rabbit  was added. After 2 h of RT incubation, ELISAs were developed with SIGMAFAST™ p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.Descriptive statistics were calculated to study various variables of importance . Statistics were performed using Graph pad, version 5  one-way ANOVA or with a two-tailed distribution and two-sample unequal variance with post hoc Tukey’s analysis. Statistical significance was defined as follows: * p ≤ 0.01) and 0.68-fold (p ≤ 0.001) at 100 mM EtOH treatment, compared to control-treated cells To determine the effect of EtOH on the HeLa cell line, the following experiments were performed. HeLa cells not treated (control) or treated with EtOH at 50 mM or 100 mM in the exosome-free medium for 24 h and 48 h were evaluated for cell viability. The trypan blue exclusion assay was performed to determine the number of viable cells present at 24 h or 48 h of EtOH administration. At 24 h, HeLa cell viability significantly decreased when EtOH was administered A. At 48 7 particles/mL  and SDS-PAGE/Western blot analyses were performed. The proteins of interest are well-known EV-associated proteins, CD81, Hsp70, and clathrin. Equal amounts of EVs from the 48 h samples  were evaluated. The data demonstrated that the exosomal marker, CD81, was found in all HeLa-derived EVs. EVs derived from HeLa cells after 48 h of treatment with 100 mM EtOH significantly expressed CD81 ** p ≤ 0.01 F. The dap ≤ 0.01 G1,2. TheRabs are a group of GTPase proteins that are involved in a variety of biogenesis-related functions including membrane trafficking, vesicle formation, and secretion. We evaluated Rab 5 and Rab 7 expression in HeLa cell lysates and HeLa derived-EVs after EtOH administration. Cell lysates at the 48 h time point were selected for evaluation. A slight decrease in Rab 5 and Rab 7 protein expression in HeLa cell lysates at 48 h of 100 mM EtOH treatment was observed A,C. EquaHsps are molecular chaperone proteins that mediate the synthesis and folding of proteins. They are induced in response to environmental stimuli and stressors such as alcohol ,44. In aFAS gene (full form) provides instructions for making a protein that is involved in cell signaling. Three FAS proteins form a trimer that then interacts with other molecules to perform its signaling function. This signaling starts a process known as the “caspase cascade”. The caspase cascade is a sequence of steps that results in the self-destruction of cells (apoptosis) when they are not needed [p ≤ 0.001, p ≤ 0.001)   B. Levels ≤ 0.05) D. These Despite a decrease in the prevalence of cervical cancer in developing countries, cervical cancer is still the second highest cancer among women . In receAlcohol consumption has been shown to increase the acquisition and pathogenesis of some viruses, such as human immunodeficiency virus ,55,56,57Our results showed that EtOH administration significantly sensitized HeLa cells, and a noticeable reduction in HeLa viability at 50 mM and 100 mM concentrations at a time point of 48 h exposure when compared to control cells . EVs werRab 5 and Rab 7 are characteristically associated with the early endosome and late endosomes. The data illustrated a slight decrease in Rab 5 and Rab 7 protein expression in cell lysates at 48 h, 100 mM EtOH treatment A,C. ThisVarious Hsps were detected in HeLa-derived EVs, particularly Hsp60 A,D. We oOf the Hsps investigated in these studies, we observed the greatest range of expressions within Hsp70. Hsp70 protects brain cells against ischemia and other stressors . One mecHsp90 has been found to be released in EVs by cancer cells ,77. Hsp9Both FAS and cleaved caspase 9  are alteOur data illustrate that EtOH modulates cellular biogenesis of HeLa cells. Specifically, EtOH modulates EV biogenesis and the cargo of HeLa derived-EV. Although HeLa cells used the primary model in this study, the research findings may have substantial implications on diagnostics and therapy for a variety of cell types and organ systems. Further investigation is needed to elucidate the mechanism(s) involved in these processes.


Invasive micropapillary carcinoma (IMPC) of the breast is characterized by its unique morphology and frequent nodal metastasis. However, the mechanism for development of this unique subtype has not been clearly elucidated. The aim of this study was to obtain a better understanding of IMPC.Using representative cases of mixed IMPC, mRNA expression in the micropapillary area and usual invasive area was compared. Then, immunohistochemical analyses for 294 cases (76 invasive carcinomas with a micropapillary feature [ICMF] and 218 invasive carcinomas without a micropapillary feature [ICNMF]) were conducted. Clinicopathological analyses were also studied.p = 0.153) or overall survival (p = 0.272) were not significant.DNA microarray analyses for mixed IMPC showed that BC-1514 (C21orf118) was commonly upregulated in the micropapillary area. CAMK2N1, CD1d, PJA2, RPL5, SAMD13, TCF4, and TXNIP were commonly downregulated in the micropapillary area. Immunohistochemically, we confirmed that BC-1514 was more upregulated in ICMF than in ICNMF. CD1d and PJA2 were more downregulated in ICMF than ICNMF. All patients with cases of PJA2 overexpression survived without cancer recurrence during the follow-up period, although the differences for disease-free (The CD1d- and PJA2-related tumour microenvironment might be crucial for IMPC. Further study of the immune microenvironment and micropapillary features is warranted. Invasive micropapillary carcinoma (IMPC) of the breast is characterized by its unique morphology; formation of micropapillae within clear spaces separated by fibrous stroma, and reverse polarity . A reverAfter approval by the Institutional Review Board of Kawasaki Medical School ethics committee , two representative cases of mixed IMPC were extracted from the database of the Department of Pathology, Kawasaki Medical School. Case 1 was ER–, PgR–, and HER2 3+, with histologic grade 2 and pT2N3aM0 Fig. . Case 2 Polyclonal antibodies were generated for SAMD13, TCF4 and TXNIP using synthetic peptides of their specific amino acid structures: positions 31–46 for SAMD13; positions 756–773 for TCF; and positions 333–351 for TXNIP. Commercially available rabbit polyclonal antibodies were used for BC-1514 , BC-1514 , CAMK2N1 , PJA2 , and RPL5 (Abcam). A commercial mouse monoclonal antibody was used for CD1D .The first tissue microarray was constructed with 231 consecutive surgical cases of invasive breast cancers from Kawasaki Medical School Hospital from September 2009 to December 2010. The first microarray contained 13 cases of invasive carcinoma with a micropapillary feature . The second tissue microarray was constructed with 63 cases of ICMF from January 2011 to December 2014. A KIN-2 system  with a 2-mm needle was used for the tissue microarray. For cases of ICMF, only the micropapillary area was sampled. ER and PgR were judged using 1% cutoff .Immunostaining was performed using an EnVision Plus kit . We cut 4 μm sections from the microarray tissue. After dewaxing and hydration, they were placed in a bath of hot citrate buffer, pH 6.0 at 95 °C for 40 min for BC-1514 (C15), BC-1514 (W12), CAMK2N1, CD1D, PJA2, SAMD13, TCF4 and TXNIP, or of Target Retrieval Solution, pH 9.0 (Dako) for RPL5 at 95 °C for 40 min. The sections were incubated with the primary antibodies for overnight at 4 °C. The dilutions of primary antibodies were: 1:200 for BC-1514 (C15), 1:100 for BC-1514 (W12), 1:500 for CAMK2N1, 1:300 for CD1D, 1:100 for PJA2, and 1:1000 for RPL5. For SAMD13, TCF4, and TXNIP, the antibodies were used at 2 μg/ml. The chromogen used was 3,3′ -diaminobenzidine tetrachloride, and the sections were counterstained with hematoxylin.Immunohistochemistry was analysed using a histoscore that was calculated by multiplying the positive area (%) and intensity . Immunohistochemical analyses were evaluated in blinded manner.p <  0.05 was considered significant.Statistical analyses were performed using IBM SPSS Statistics for Windows . The commonly up- and downregulated factors in IMPC compared with ICNST by DNA microarray are shown in Table The immunohistochemical expression of BC-1514 (C15), BC-1514 (W12), CAMK2N1, CD1d, PJA2, RPL5, SAMD13, TCF4, and TXNIP are summarized in Table p = 0.373). However, expression of CD1d by ICMF is lower than that of normal breast tissue (p = 0.008). The expression of PJA2 by ICNMF is higher than that of normal breast tissue (p <  0.001). The expression of PJA2 by ICMF is similar to that of normal breast tissue (p = 0.259).The expression of CD1d by ICNMF is similar to that of normal breast tissue (p = 0.015). Histological tumour grade and HER2 showed positive correlations with BC-1514 (W12), respectively (p = 0.009 and p = 0.043). The cases with a high Ki-67 index showed higher BC-1514 (C15) (p = 0.010), BC-1514 (W12) (p = 0.011), and CD1d (p = 0.036) than the cases with a low Ki-67 index.The clinicopathological analyses of BC-1514 (C15), BC-1514 (W12), CD1d, and PJA2 are shown in Table p = 0.001, < 0.001 and 0.011). pT, pN, stage, histological grade, and PgR were significant for overall survival . BC-1514 , CD1d, and PJA2 did not show any significant difference for disease-free survival or overall survival. All patients with cases having high PJA2 survived without cancer recurrence (19 of 19) using the third quadrant as a cut-off value, but there was no significant difference .The median follow-up periods for disease-free survival was 87.8 months  and for overall survival was 88.2 months . The univariate Cox hazard survival analyses are shown in Table CD1d-presents lipid antigens to activate natural killer T (NKT) cells, through the interaction with the T-cell receptor present on NKT membranes . NKT celPJA2, also known as PRAJA2, regulates the protein kinase A signal strength and duration in response to cAMP . PJA2 inCD1d, PJA2, and granzyme A precursor also showed lower expression in IMPC than in ICNST in our DNA microarray screening. Granzyme A, an enzyme present in cytotoxic T lymphocytes, has tumouricidal activity , 25. TheHomo sapiens has been withdrawn by the National Center for Biotechnology Information because of insufficient evidence . Our immunohistochemistry for BC-1514 probably showed some cross reaction to an unknown substance.We demonstrated BC-1514 mRNA upregulation in IMPC and greater immunohistochemical expression in ICMF than ICNMF. However, the record of BC-1514 for One of the limitations of this study is that our DNA microarray data was obtained from only two mixed IMPC cases. We thought that the candidate markers for micropapillary feature were well narrowed down by just two cases. However, four  of eight markers showed contradictory results, and TXNIP did not show a significant difference by immunohistochemistry. Additional DNA microarray would be promising to increase the accuracy, and should have improved the efficiency of our immunohistochemical study. Our DNA microarray data should be interpreted with caution.Our present study suggests the CD1d- and PJA2-related tumour microenvironment might be crucial for IMPC. Further study of the immune microenvironment and micropapillary feature is warranted.


Total AR content and liquid chromatography/mass spectrometry (LC-MS)-based profiles were recorded for each extract, in addition to their antifungal activity, to help integrate these chemical and biological datasets by orthogonal partial least squares regression. In this study, we developed and used a micro-scale amended medium (MSAM) assay to evaluate the in vitro mycelial growth inhibition at low amounts of extracts. Triticale husk-derived extracts had the highest AR content , exhibiting >79% inhibition at the highest doses (10.0–1.0 µg/µL). Correlation of the chemical and antifungal datasets using supervised metabolite profiling revealed that 5-n-nonadecanylresorcinol, 5-n-heneicosylresorcinol, and 5-n-tricosyl-resorcinol were the most active ARs occurring in cereal products from Colombia. Hence, we propose the biochemometrics-based approach as a useful tool for identifying AR-like antifungals against F. oxysporum.A rapid and convenient biochemometrics-based analysis of several cereal-derived extracts was used to identify  Cereals comprise some plant materials commonly called grasses—a merged name to designate those monocots of the Poaceae family. This family includes rice, wheat, rye, barley, oat, and other cereals, which represent the main source of food worldwide. Species within this family mostly produce a fruit called caryopsis . According to the Food and Agriculture Organization (FAO), 2610 million tons of cereals were produced in 2018, with wheat being the most prominent (754.1 million tons) − adducts that resulted from low-resolution (LR) and high-resolution (HR) ESIMS analyses, ensured a correct assignment through molecular ions and exact mass in agreement with the molecular formula for the identified metabolites (error < 4 ppm). Alkenylresorcinols such as 1, 4, 6, and 11 exhibited ions in HRESIMS spectra, involving losses in the side chain by α-cleavage at double bonds . The resorcinol moiety of compounds 1–12 was also established after derivatization using N-methyl-N-trimethylsilyl-trifluoroacetamide activated II® (MSTFA), following the protocol previously described +), as well as fragments at m/z 281, 310, and 341, which are very common for ARs [O-silylated derivative of compounds 1–12 was consequently detected ([M + (TMS)2]+). Differences between the fragment at m/z 268 and molecular ion, indicated the particular length for the side chain, which in turn involved consecutive losses of CH2 fragments −) was present in samples with lower activity, indicating no relationship with antifungal action. In contrast, a compound marked as P , the fungicidal ingredient used to protect the certified oat caryopsis. This reference material with this fungicide were used as internal standards and, as observed in the respective S-line  with LC-ESIMS (independent) data as a tool for identifying antifungal components, the subsequent biochemometrics-based analysis through the internal cross-validated construction of a single-LS model c evidencca. 80%) b. These 3.7 min, d was idee S-line d, this cFusarium oxysporum strain was obtained from a Cape gooseberry  crop, preserved on Whatman paper # 1 at −20 °C, and reactivated in Potato Dextrose Agar (PDA) at full concentration before use.Plant materials of some cereals were obtained from several places in Colombia. Caryopsis and husk of barley, triticale, yellow corn, and wheat were gathered from an experimental crop at Nueva Granada Campus in Cajicá, Colombia; caryopsis and husk of rice, oat, pearl barley, soy, and sorghum, wheat flour, corn  flour, and oat flakes were purchased from different marketplaces in Bogotá, Colombia. All aforementioned materials were ensured to be free of pesticide traces based on the organic cultivation protocol implemented for human/animal consumption. In addition, certificate oat material (husk and caryopsis), previously treated with fludioxonil, was also purchased from a marketplace in Bogotá, Colombia for use as reference material. The n-hexane, ethyl acetate, isopropanol, acetone, and methanol). The resulting mixture was then filtered and concentrated under reduced pressure to obtain the unfractionated, crude extract in each solvent. All kernel samples were grinded in a Waring WGS30 grinder machine , and the husk was separated from the flour using a stainless steel sifter  prior to the extraction. The best AR extraction solvent was determined as follows: a 24-h maceration extraction was carried out on wheat bran (1 g) with different solvents  with some modifications. Briefly, a working solution was prepared at 1:5 ratio with methanol (stock 0.5 mg/mL FBRR®), and a solution to 5 mg/mL of the samples previously prepared. An aliquot of extract solution (20 µL) was added, FBRR® working solution (2 mL) and 10% sodium carbonate  were dispensed into spectrophotometric cells. These cells were incubated for 20 min at room temperature and the absorbance was measured on a Genesys 20 spectrophotometer  at 480 nm. Total AR content was then calculated from a previously constructed calibration curve using olivetol (5-pentylresorcinol) as standard. The selected solvent  was allowed to remove AR from other samples using 60 g of plant material for the respective extract preparation. Thus, the procedure described previously for wheat bran was therefore performed for all samples. TARC values were expressed as µg olivetol equivalent per g dried extract (µg OE/g DE).Total AR content (TARC) was determined by the colorimetric quantification method reported by Sampietro et al. , using tOnce the extraction procedure was standardized/optimized as described in ® column  was used for analysis at 0.6 mL/min using the mixtures MeOH:H2O (8:2) (A) and isopropanol:MeOH (3:7) (B) in a gradient method . Electrospray ionization interface (ESI) was operated in a negative ion mode (scan 100–2000 m/z), desolvation line temperature at 250 °C, nitrogen as nebulizer gas at 1.5 L/min, drying gas at 8 L/min, quadrupole energy at 7.0 eV, and collision energy 14 eV.All extracts were analyzed using Reverse Phase Ultra-Fast Liquid Chromatography coupled to a microTOFQ II mass spectrometer . A Kinetex® column  was used for analysis at 0.6 mL/min using the mixtures MeOH:H2O (8:2) (A) and isopropanol:MeOH (3:7) (B) in a gradient method ; detection was performed at 270 nm. Electrospray ionization interface (ESI) was operated simultaneously in positive and negative ion modes (scan 100–2000 m/z), desolvation line temperature at 250 °C, nitrogen as nebulizer gas at 1.5 L/min, drying gas at 8 L/min, and detector voltage at 1.4 kV.Extracts were also analyzed by Reverse Phase Ultra-Fast Liquid Chromatography coupled to a Diode Array Detector (UFLC-DAD) on a Prominence system , coupled to a Shimadzu LC2020 mass spectrometerA KinetexN-methyl-N-trimethylsilyltrifluoroacetamide activated II® . For each sample (5 mg/mL), 200 µL were dried into a chromatography vial with and insert; 20 µL of the derivatization agent were then added and incubated at 60 °C for one hour [® 5 Sil MS, Restek, State College, PA, USA) using helium (99.999%), with a flow of 1.5 mL/min, 1 µL of sample was injected in splitless mode, and the temperature of the injection port was 300 °C. The temperature program started with an initial temperature of 100 °C maintained for 1 min, then increasing from 100 °C to 250 °C at 15 °C/min, and holding for 5 min. It was then increased to 310 °C at 10 °C/min, and maintained for 30 min. The ion source and transference line temperatures were 250 °C and 310 °C, respectively.Raw extracts were derivatized with one hour . The anaF. oxysporum was performed using an innovative, micro scale amended protocol using 12-well glass plates. A previously stirred mixture (150 µL) containing Potato Dextrose Broth PDB (25.0 g/L) and bacteriological agar (0.5%) amended with extracts  was placed into each well as culture medium. The required amount of extracts to reach the final concentration was added to the warm medium, and the mixture was vigorously stirred using a vortex for 3 min until adequate dispersion was achieved, which resulted in the amended media. A 1-mm agar-mycelial plug from actively growing F. oxysporum cultures was inoculated onto the center of each well. Each test consisted of a randomized design with three replicates compared to a plate control .In vitro evaluation of mycelial growth inhibition against ® SZM-LED2, Ponteranica, Italy) and analyzed using ImageJ software . The inhibition percentage was calculated based on the area measured by ImageJ, compared to the control area, according to Equation (1): The plates were then photographed in a stereoscope (Optikap < 0.05) using R project software version 3.0.2 . In addition, the AR contents, ASCII-formatted LC-MS-derived data , and growth inhibition percentages against F. oxysporum were used to build the dataset. The resulting matrix was used for multivariate statistics (biochemometrics) through Single-Y orthogonal partial least squares (OPLS) regression using the SIMCA 13.0 software .Total contents and antifungal activity data were subjected to the Shapiro–Wilks test, parametric analysis of variance (ANOVA), and Tukey’s test  protocol, which allowed us to evaluate small amounts of cereals-derived extracts. This combination  led to the identification of bioactive AR without the need for multiple bioactivity-guided isolation steps, thus improving the efficiency and productivity of antifungal discovery purposes. Therefore, multivariate statistical modeling (based on single-Y OPLS) revealed three ARs from a set of extracts that were responsible for the observed mycelial growth inhibition such as compounds 5, 8, and 10, which were also correlated by OPLS to the AR-rich mixtures. The results therefore indicate that these compounds, abundant in cereals, are important and valuable chemical entities that could eventually be included into advance experiments focused on F. oxysporum control. Further studies  are also required for isolating these compounds and to further validate our findings.AR-rich extracts from cereals showed inhibitory activity against


We show that combining deep sequencing of stimulated memory B cell repertoires with retrieving single antigen-specific cells is a promising approach in evaluating the latent, functional B cell memory in PBMCs.Phenotypic screening of antigen-specific antibodies in human blood is a common diagnostic test for infectious agents and a correlate of protection after vaccination. In addition to long-lived antibody secreting plasma cells residing in the bone marrow, memory B cells are a latent source of antigen-experienced, long-term immunity that can be found at low frequencies in circulating peripheral blood mononuclear cells (PBMCs). Assessing the genotype, clonal frequency, quality, and function of antibodies resulting from an individual's persistent memory B cell repertoire can help inform the success or failure of immune protection. Using  Furthermore, broadly cross-reactive binding and neutralizing recombinant antibodies directed to Influenza A and HCMV from polyclonally stimulated PBMCs have been cloned from single B cells using this method  from single memory B cells isolated by FACS with the hemagglutinin trimer of influenza A H1N1. Members of these mAbs clonal lineages were identified in PBMCs vs. stimulated PBMCs repertoires, quantified, and tracked over the three blood draws. An increased sensitivity for detection of individual mAbs along with larger, branching clonal families were obtained using data from stimulated PBMCs. Overall, our data support the application of stimulating PBMC samples to gain a deeper understanding of antigen-specific circulating memory B cell repertoires.Herein, we show the benefit of combining NGS with ++/Mg++), after centrifuging at 400 × g, 20°C, for 50 min . PBMCs from each 50 ml tube were washed by diluting three times with 50 ml ambient temperature RPMI 1640 and centrifuged 200 × g, 20°C, 10 min. PBMCs were cryopreserved in 90% heat-inactivated FBS, 10% DMSO at 1 × 107 cells per vial.Leukapheresis was performed on three normal donors using Institutional Review Board (IRB)-approved consent forms and protocols by StemCell Technologies . Approximately two to three blood volumes were processed using the Spectra Optia® Apheresis System to produce a full-sized Leuko Pak. Acid citrate dextrose, solution A (ACDA) was the anticoagulant. Donors included: 536, African American age 33, male, 82 kg, 180 cm, smoker, collected 11/15/2017, 5/24/2018, and 08/27/2018; 147, Caucasian female age 21, 56 kg, 153 cm, non-smoker, collected 08/21/2018; and 682, Hispanic male age 30, 74 kg, 181 cm, smoker, collected 08/16/2018. PBMCs were further enriched from the buffy coats of 50 ml tubes with 25 ml sterile Histopaque 1.077  layered with 25 ml leukocytes diluted 1:1 in PBS (no CaHuman fibroblast cell line MRC-5 (CCL-171) was obtained from ATCC®  and grown in B cell growth media containing Corning® DMEM [+] 4.5 g/L glucose, sodium pyruvate [–] L-glutamine , 1xPen/Strep/Glu and 10% ultralow IgG HI-FBS , to 80% confluence before being treated for 4 h with 5 μg/ml mitomycin C . Monolayers of growth arrested cells were washed 3 times with PBS, harvested with trypsin, neutralized with growth media, washed 1x in growth media and finally cryopreserved using 10% DMSO, 30% HI-FBS in growth media.PBMC vials were thawed rapidly in a 37°C water bath, immediately diluted into 10 ml of B cell growth media, and pelleted at 350 × g for 5 min. The sample was resuspended in growth media and B cells were negatively selected using the EasySep Human B cell Isolation Kit according to the manufacturer's protocol . Because our samples contained greater than one million PBMCs, B cell enrichment was used to provide a corresponding enrichment of B cell RNA in the total RNA added to the reverse transcription reaction. The final cells from negative enrichment were pelleted at 350 × g for 5 min, resuspended and lysed in Qiagen RLT buffer with beta-mercaptoethanol for 10 min, frozen on dry ice, and transferred to −80°C storage until RNA purification with the Qiagen AllPrep RNA/DNA kit .6 feeder cells were seeded in a T25 flask (VWR) in 4 ml of B cell growth media and cultured overnight in a humidified 37°C, 5% CO2 incubator. PBMCs were quickly thawed at 37°C, washed 1x in 10 ml of growth media, and resuspended in 4 ml of 2x B cell growth media containing 2 × ITS from 100x Insulin, Transferrin, Selenium (Thermo Fisher Scientific), 20 ng/ml IL-10, 2 ng/ml IL-2, 10 ng/ml IL-15, 10 ng/ml IL-6  and 4 μg/ml CpG . The 4 ml of PBMCs were then added to the T25 flask with 4 ml of conditioned feeder cell media. The final 8 ml cell culture was allowed to grow for 5 days at 37°C, 5% CO2 in a humidified incubator. At day 5 the cells were pelleted at 350 × g for 5 min, resuspended and lysed in Qiagen RLT buffer with beta-mercaptoethanol for 10 min, frozen on dry ice, and transferred to −80°C storage until RNA purification. Since the culture conditions specifically expand memory B cells at the expense of viability of other cell types, no B cell enrichment was utilized at this step. Supernatants were also harvested to assay total IgG, IgA, and IgM secretion by indirect ELISA, as described below.For generating stimulated PBMC samples, we applied methods previously published by McCutcheon et al.  to clone96-well, Nunc Maxisorp™ plates  were coated overnight with anti-human IgG Fc , anti-human IgA Fc or anti-human IgM Fc  at 2 μg/ml in PBS, pH7.2. The next day the plate was washed 3x 300 μl PBST and blocked for 1 h in 1% BSA/PBS. A standard curve was prepared using human reference serum (Bethyl Labs) in 1/3 dilutions starting from targeted serum dilutions to give 200 ng/ml of IgG, IgA, or IgM, in assay diluent (0.5% BSA//PBS/0.05% Tween-20). Supernatants from stimulated PBMCs were diluted in assay diluent between 1/5 and 1/100. Both standards and samples were allowed to bind for 2 h, washed 6x 300 μl PBST and a 1/5000 cocktail of anti-human kappa-HRP and lambda-HRP antibodies  added for 1 h in assay diluent. After 6x 300 μl PBST washes, the plate was developed with KPL SureBlue™ TMB (VWR).n = 12): lambda (n = 16) forward primers. Kappa RT and forward primer pools were prepared by mixing 10 μl of each kappa RT (n = 2) primer or kappa forward (n = 8) primer from the individual 100 μM stocks and then diluting the mix to 10 μM final.Dry oligos of desalted purity (IDT) were reconstituted at 100 μM in Qiagen EB and stored in aliquots at −80°C. The oligos are shown in Total RNA yields from the PBMC and stimulated PBMCs were in the range of 3 micrograms (50–100 ng/μl in 30 μl Qiagen EB buffer) determined by absorbance at 260 nm on the NanoDrop™ One (Thermo Fisher Scientific). An input of 100–200 ng total RNA was used for first strand cDNA synthesis with gene-specific reverse transcription (RT) primers directed to the constant regions. The RT primers for IgG, IgM, IgA, and lambda were pooled, whereas the kappa RT was done in a separate reaction. Light chain kappa RT was carried out in separate reactions because the transcript abundance and amplification efficiency tended to out-compete heavy and lambda chains in multiplexed reactions. Primers are shown in https://immcantation.readthedocs.io. Because the Immcantation pipeline can be run using Docker containers, we created a cloud-based workflow incorporating Reflow (https://github.com/grailbio/reflow) that allowed for seamless processing of the constituent Immcantation software packages. The workflow is available at Github (https://github.com/czbiohub/bcell_pipeline). Some key characteristics of our workflow include the use of unique molecular identifiers  at  at httpss , we detin vivo antibody molecular evolution to a consensus directed against common epitopes of vaccines or disease. As the field gains more and more knowledge on immune repertoires and collects data on responses to disease and vaccination, we will get closer to being able to deliberately direct our immune system toward epitopes yielding positive health outcomes.Clonal expansions of B cells occur in response to specific immune stimulation and evidence for a corresponding convergence of germline gene arrangements has been shown e.g., –27]. Clo. Clo27)]https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA524904 and https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA526542.The datasets generated for this study can be found in the NCBI Sequence Read Archive (SRA) under accession PRJNA524904 (MiSeq data) and PRJNA526542 (transcriptomic data) at KM conceived and designed the study. EW, NF, and KM carried out the experiments. EW and KM performed data analysis and wrote the manuscript. EW and AM developed the sequence analysis pipeline. PK provided study oversight and review. All the authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.