Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents.Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.We have studied clinical  Cystic fibrosis (CF) is the result of mutations in the CF transmembrane conductance regulator affecting epithelial chloride and bicarbonate transport. One result is development of thick respiratory secretions, which results in airway obstruction and recurrent episodes of lung inflammation and infection, leading to acute and chronic deterioration of lung function and a shortened lifespan . The affPseudomonas aeruginosa (Pa) [Aspergillus fumigatus (Af) [Pa evolves in CF airways, producing variants, such as those resulting in mucoid colony types, which are adapted to chronic residence there [Af is ubiquitous in ambient air and the environment, and thus can be inhaled and subsequently establish residency. Both organisms are proficient adapters to environmental stress and relatively resistant to current antimicrobials. They are suspected as important agents in promoting mucus plug formation in the airways, and both are known to form biofilms in vitro and in vivo [Af [Pa [Af can cause allergic bronchopulmonary aspergillosis in up to 15% of CF patients, a complication that causes repeated acute exacerbations, institution of immunosuppressive therapy, and accelerated decline in lung function [Pa, Af also produces secondary metabolites, in the environment as well as in vivo, which are known tissue toxins and also have immunosuppressive actions on the host response [Pa or Af has been associated with a more rapid decline in CF pulmonary function [The most common bacterium and fungus infecting these airways are osa (Pa)  and Aspetus (Af) –7, respece there ,8,9. Af  in vivo ,9–20. Mi in vivo . The attvivo [Af  and algi [Af [Pa . In addifunction . As is tresponse . Infectifunction ,25–33, wfunction ,34. Bothfunction –39 or otfunction ,41, in lPa-secreted molecules have been well studied for their antifungal activities, a property that has been demonstrated in vitro [Pa isolates, none representatives of variants that establish chronic residency in CF airways. Moreover, Pa-associated factors involved in this inter-kingdom inhibition continue to be elucidated [Pa phenotypic variants obtained from CF and non-CF patients on Af biofilm formation and preformed Af biofilm.It is therefore important to study the interactions between these two pathogens. in vitro –47. Thesucidated . The aimPa recovered from non-CF patients (n = 16 isolates), or CF patient sputum (n = 10), from Stanford University Hospital and clinics were evaluated. Among the CF isolates, five were mucoid colony phenotype variants [Pa isolate obtained from the same CF patient the same day, 2 non-mucoid Pa isolates from another CF patient 6 mos. apart, and 2 Pa isolates with different colonial morphologies from each of 2 non-CF patients obtained the same day, plus another Pa isolate from one of these patients one month later. Af isolate 10AF, a virulent non-CF patient isolate [Af isolate throughout this study. Nine sputum Af isolates, also identified by molecular methods to be Af sensu stricto [Any CF isolates from patient respiratory cultures were obtained after written informed consent, for biobanking of the patients’ specimens and subsequent use, approved by the Stanford Institutional Review Board. Other isolates were obtained following clinically indicated cultures. Twenty-six clinical isolates of variants ,8,9 and  isolate ,50, was  stricto , were ob stricto  and addiAf conidia were obtained as follows: Af was taken from stock suspensions stored at -80°C and then grown for 4 days on Sabouraud Dextrose Agar  at 37°C. Conidia were harvested by gently washing with 0.05% Tween-80  in 0.9% saline .Pa stocks were maintained at -80°C in Microbank microbial storage vials . Each frozen Pa stock culture was initially inoculated onto Trypticase Soy + 5% sheep blood agar plates  and incubated overnight at 37°C. Pa colonies were then picked, 1–2 loopfuls of the bacteria diluted in RPMI-1640 medium, and the suspension adjusted in the spectrophotometer at A610 to an absorbance of 0.35–0.40 with fresh RPMI-1640 medium. This corresponded to 109/ml, with variation over a 3-fold range. A 1:30 dilution was then made in studies of direct Pa action on Af biofilm, or production of Pa culture supernatants, to be described. We found, in the studies to be described, that neither direct Pa action, nor Pa supernatant action, on Af biofilm was particularly sensitive to the size of the initial Pa inoculum in those studies, as variations of at least 10-fold in live Pa cells, and at least 33-fold in the inoculum used to make planktonic Pa supernatants, resulted in no significant differences in activity.Inhibition of growth was assessed by using a conidial inoculum, following guidelines for susceptibility testing of filamentous organisms .in vitro model described previously [Af biofilm, sterile polystyrene disks  were placed in 12-well tissue culture plates . Each well contained 3 ml of fresh RPMI-1640 medium  with 105Af conidia/ml.Biofilms were formed by using a modified eviously . To formDisks were incubated at 37°C for 16 h with shaking at 70 rpm, to allow the fungal cells to attach. Following the attachment phase, disks were gently rinsed in sterile saline , transferred to new plates containing fresh RPMI-1640 medium, and incubated for an additional 24 h at 37°C with shaking at 100 rpm . Biofilm5Af /well always involved in biofilm formation, the ratio of Pa to Af was 3.3 x 107:3 x 105/well. Disks were then rinsed gently with sterile saline, transferred to new plates containing fresh RPMI-1640 medium, and incubated for an additional 24 h at 37°C with shaking at 70 rpm  and bacterial cells (0.1 ml), prepared as described above, were combined in 12-well tissue culture plates in fresh RPMI-1640 medium and incubated at 37°C for 16 h with shaking at 70 rpm. Thus, for the 3 x 10t 70 rpm .Pa suspension (3 ml containing 3.3 x 107 cells) prepared as described above, and incubated for an additional 24 h at 37°C with shaking at 70 rpm  for 24 h at 37°C with shaking at 70 rpm. The spent medium was removed, transferred to a new 50 ml conical tube, and centrifuged for 30 min at 2,000 x g to remove any suspended cells or debris. The planktonic supernatant was gently removed, filter sterilized (0.22 μm)  and used. Filtered supernatant (1.5 ml) was added to selected wells of a 12-well tissue culture plate previously inoculated with 1.5 ml of the standardized Af suspension in fresh RPMI-1640. Fungal biofilms attached and formed as described above. Disks containing biofilms were washed 3 times with sterile saline and transferred to a new plate containing fresh RPMI-1640, and incubated for an additional 24 h at 37°C with shaking at 70 rpm  for 2 h at 37°C on a 100 rpm shaker incubator (attachment phase). The liquid was then removed and the flasks were rinsed gently 3 times with sterile saline. Twenty ml of fresh RPMI-1640 was added to the flask and adhered cells formed Pa biofilms for 22 h , observed by the presence of a layer of bacterial growth on the inner surface of the flask. The spent medium was then removed and processed as described above. Af conidia forming biofilms or preformed Af biofilms were challenged with the Pa biofilm culture filtrate, as described above. Bacterial supernatant-free wells were also included as controls  was added to the RPMI-1640 medium at each step to produce a 10% concentration.For sets of experiments with Af biofilm was studied, at the end of the challenge period with either live Pa cells or filtered Pa cultured supernatants (or control media), the Af biofilm was studied with the XTT assay. The tetrazolium salt, XTT   was used to measure the metabolic activity of Af. XTT is reduced by a mitochondrial dehydrogenase to a water-soluble formazan product. Af biofilm discs were rinsed 3 times in sterile saline and transferred to fresh wells containing 3 ml sterile phosphate-buffered saline (PBS) . Menadione (Sigma) 0.85 gm was added to 5 ml acetone, and mixed in a 1:11 ratio of menadione to XTT (1 mg/ml). The XTT-menadione solution (180 μl) was added to each well and the plates were incubated in the dark for 2 h at 37°C. Following incubation, the contents of the wells were collected and centrifuged for 10 min at 13,300 x g. The pellet was then discarded and the absorbance at 490 nm of the supernatant was determined with a spectrophotometer . Although XTT is a measure of metabolic activity of cells, previous studies of Af have indicated XTT results are linear with mass, and equated XTT result with dry weight [Either when conidia formed biofilm, or when preformed y weight –56.Pa cells co-incubated with Af, we determined that the XTT readings only reflected the Af by studying Pa similarly cultured without Af, showing there was no increase of XTT reduction compared to background without any microbes present.For the assay of live Af biofilms were formed on disks as described above. The biofilms were challenged with Pa supernatants obtained from a representative of each of the 3 Pa groups. After incubation at 37°C, the disks were washed three times in sterile PBS and stained using a fluorescent stain , prepared according to the manufacturer’s instructions. FUN-1 (1 μl) from a 10 mM stock was mixed in 1 ml of PBS. Staining was performed as previously described [Af biofilm, a bright green cytoplasmic stain produced after passive diffusion. An additional analysis was made to visualize the effect of bacterial supernatant on the fungal biofilm metabolic activity. A red fluorescence, visualized from intravacuolar staining, was noted, which requires both plasma membrane integrity and metabolic capability (data not shown). Dead cells would have fluoresced bright yellow-green, with no red structures, and were not apparent in our images.escribed ,13. ThreSections on the xy plane were taken at 1 μm intervals along the z-axis to determine the depth of the biofilms. Microscopic visualization and image acquisition of biofilms were conducted at the Stanford Biofilm Research Center using an upright Leica TCDSP2 scanning confocal laser microscope  equipped with an argon/krypton laser and detectors, and filter sets for monitoring of green  and red . Images were obtained using a 63 x 1.4 Plan-APOChromat DIC  objectives. Multichannel simulated fluorescence projection  images and vertical cross sections through the biofilm were generated using the IMARIS software package . Images were processed for display by using Photoshop software . Representative images were taken.Pa groups. Pa planktonic and biofilm culture filtrates were obtained as described above. The culture filtrates or sterile distilled water were diluted in fresh RPMI supplemented with 10% FBS, so that the percent of fresh medium in each test situation was decreased as the spent medium or sterile distilled water increased. Af biofilms were formed in this mixed media, as described above, or planktonic Af growth was studied [A dose response study was performed using one isolate from each of the 3  studied .Pa planktonic and biofilm culture filtrates were prepared as described above. In brief, the final cell-free supernatants were (a) heat treated at 56°C for 30 min and immediately used or (b) frozen at -80°C, stored for 6 days, and thawed. These filtrates were then tested compared to fresh filtrates against either Af conidia forming biofilm, or preformed biofilm, as described above.The Pa or phage DNA or a Pa protein(s) were involved in the inhibition of Af, we studied the effect of pretreatment of Pa culture filtrates with DNase I  or Proteinase K (Sigma) and measured the filtrate inhibitory activity on Af biofilm formation  (Sigma) was used to inactivate the DNase I activity. Experiments with DNase I treatment involved growing the Af for 16 hours in the presence of (a) Pa filtrate, (b) DNase I  + Pa filtrate, (c) DNase I + EDTA  + Pa filtrate, (d) EDTA + Pa filtrate, or (e) EDTA alone or (f) DNase + EDTA (in fresh RPMI medium). These were followed by 24 h growth in fresh RPMI-1640 alone.Pa filtrate, (b) Proteinase K  + Pa filtrate, or (c) medium treated with Proteinase K.For Proteinase K pretreatment, conidia were incubated with (a) A 30,000 MW cutoff filter was obtained from Millipore Ltd., Tullagreen, Ireland.Pa isolates was assessed as previously described [Elastolytic production of escribed .3 was obtained from Sigma.FeCln is not stated, each experiment was performed at least two times, with triplicate wells each time. Data from experiments using XTT as the assay parameter were collected in blocks. Each block was comprised of n = 6 values derived from of two sets of triplicate values collected on different days. For the data presented in Figs Pa, mucoid Pa and nonmucoid Pa (Pa was either live cells or spent culture filtrates as indicated). Each block of four conditions was analyzed separately using one-way analysis of variance (ANOVA) followed by a Tukey’s post-hoc test to adjust for the multiple comparisons being performed. Prior to pooling, the data in all blocks were verified to be normally distributed and to have homogenous variances. An initial ANOVA comparison of the five blocks for each condition  showed the blocks for that condition  not be significantly different. These results allowed us to pool the data for each condition with a result of n = 30 each. The pooled data were analyzed using one-way ANOVA with a Tukey’s post-test. For comparisons across live cell versus planktonic or biolfilm supernatants, control values were first compared and determined to be no different, which was followed by ANOVA and Tukey’s comparisons of the experimental sets. All statistical analyses were done using GraphPad Prism . Statistical significance was considered P<0.05.Where Pa from CF or non-CF patients showed differences in their interactions with Af. Thus we designed studies to assess Pa from non-CF patients compared with mucoid and nonmucoid Pa from CF patients. Two experimental designs were used. The first was that of the effects of Pa on the development of biofilm by Af conidia and the second was that of the effects of Pa on preformed Af biofilm. In all of these studies we assessed the effects of live Pa cells and the effects of spent culture filtrates from Pa planktonic or biofilm growth and initially relied on the parameter of metabolic activity of Af, based on XTT reduction. For studies in Figs A summary of all the studies to now be described is in Af conidia to live Pa cells or filtrates for 16 h followed by an additional 24 h of biofilm growth before metabolic assay of XTT reduction.The experimental design is shown in Figs Pa cells during Af biofilm formation, as assessed by metabolic activity. Comparisons of effect of individual isolates of Pa on Af with their respective Af control showed that each isolate significantly inhibited Af during the formation of Af biofilm . Pooling of the 5 blocks of data showed that all three groups of Pa isolates significantly inhibited fungal biofilm formation . Of interest, these data indicate that the inhibitory effect of CF mucoid Pa or CF non-mucoid isolates was higher compared to the effect of the non-CF isolates , and CF non-mucoid cells were more inhibitory than CF mucoid cells (P<0.001).in vitro activity of culture filtrates from Pa grown under planktonic conditions on the fungal biofilm formation, as assessed by metabolic activity. Comparisons showed that the planktonic culture filtrates of each individual isolate of Pa significantly inhibited Af during biofilm formation in comparison with controls (P<0.001). Similarly, the pooled data showed that the planktonic culture filtrates of each of the three groups of Pa isolates significantly inhibited Af . The inhibitory effect on Af biofilm formation by the culture filtrates from CF mucoid or CF non-mucoid Pa isolates was higher compared to the effect of the non-CF filtrates , and culture filtrates from CF non-mucoid Pa cells were more inhibitory than those from CF mucoid cells (P< 0.001).Pa isolates grown under biofilm conditions are shown in P<0.001, all comparisons). Examination of the pooled dated showed that biofim culture filtrates of Pa isolates from CF or non-CF patients significantly inhibited the metabolic activity of Af in biofilm development compared to the controls . In addition, culture filtrates from CF mucoid or non-mucoid Pa isolates were significantly more inhibitory than those from non-CF isolates ; CF non-mucoid Pa isolates biofilm culture filtrates were more inhibitory than those from mucoid isolates (P<0.001).The results of incubation of the conidia with culture filtrates obtained from Af biofilm to be described, we cannot completely rule out that differences in Af biofilm formation are a result of growth inhibition of the fungus. Studies (described below) of Pa inhibition of, by contrast, purely planktonic growth of Af, where there were not significant differences between the three Pa groupings, suggest the inhibitory differences of Pa groups relates more to the effect on Af biofilm formation.Whereas biofilm formation appears inhibited in the preceding studies, and in CLSM studies and studies of preformed Af to form biofilm for 16 h and then exposing this biofilm to live Pa or culture filtrates for an additional 24 h before XTT assessment of Af metabolic activity. Assessment of the inhibitory effects of the live Pa cells showed that only a single non-CF isolate, Pa19, significantly inhibited the preformed Af biofilm (P<0.01), whereas the other four non-CF isolates had no significant effect (P>0.05). Compared to preformed Af biofilm controls, all isolates of non-mucoid or mucoid live Pa from CF patients significantly inhibited the metabolic activity of preformed Af biofilm (P<0.001). Pooling of the five blocks of data showed that non-CF isolates were not inhibitory, whereas mucoid Pa isolates and non-mucoid CF isolates significantly inhibited the metabolic activity of the preformed Af biofilm in comparisons to controls or non-CF isolates of Pa . Non-mucoid CF isolates were more inhibitory than mucoid CF isolates (P<0.001)  .Pa culture filtrates on preformed Af biofilm. Comparisons of individual culture filtrate activity from individual isolates showed that a single non-CF isolate, Pa19, filtrate was inhibitory (P<0.05), whereas all those from individual filtrates from non-mucoid or mucoid Pa from CF patients, grown under planktonic conditions, significantly inhibited the oxidative metabolism of preformed Af biofilm . The results of analysis of the pooled data showed non-CF planktonic culture filtrates had no inhibitory activity, whereas planktonic filtrates from mucoid or non-mucoid Pa isolates were more inhibitory than controls or filtrates from non-CF isolates . Non-mucoid CF isolate filtrates were more inhibitory than mucoid CF filtrates (P<0.001).Pa isolates had on preformed Af biofilm. No inhibitory activity was exhibited by the non-CF biofilm filtrates (P>0.05), whereas all biofilm filtrates from mucoid or non-mucoid CF Pa from CF patients significantly inhibited preformed Af biofilm, compared to preformed Af biofilm controls (P<0.001). Similarly, the comparisons of the pooled data showed that non-CF isolate biofilm filtrates were not inhibitory and both mucoid and non-mucoid isolate biofilm filtrates were more inhibitory than controls or filtrates of non-CF isolates . Again, non-mucoid CF isolate biofilm filtrates were more inhibitory than those of mucoid CF isolates (P<0.001).Pa group (non-CF (Pa19), non-mucoid CF (Pa10) and mucoid CF (Pa11)))Figs  and 3. E11))Figs . A 106 iPa isolates were used, as well as the mucoid and non-mucoid CF isolates.Because components of serum can also be present where there is local inflammation, such as in the airways, to more closely mimic in vivo conditions it is of interest to know whether the interactions described above might be different in the presence of serum. For these studies, serum was added to the medium in all steps , as indiPa live mucoid cells, or the filtrates of such cultures grown planktonically or as Pa biofilm, were, with serum present, not statistically significantly inhibitory, as assessed metabolically, to preformed Af biofilm. Specifically, absent that one exception, the significance and conclusions with (a) mucoid Pa and Af biofilm formation, or (b) non-CF Pa or non-mucoid CF Pa, as live cells or culture supernatants , were the same as the results in the absence of serum, when tested against either (c) Af biofilm formation or (d) preformed Af biofilm, Again, non-mucoid Pa isolates were most inhibitory in all conditions, and preformed Af biofilm the most resistant. None of the 16 non-CF Pa isolates behaved differently than the rest of that group (data not shown).Overall, the results were virtually the same as those from the studies done in the absence of serum. However, there were exceptions. The Pa isolates, as described in the preceding section, from each Pa group), we omitted serum from Pa while generating supernatants, but added serum during Af biofilm formation or preformed Af biofilm development. We found the same results, as described, as when serum was present or absent in all phases.In additional experiments . The supernatants of non-mucoid CF Pa were inhibitory to both Af biofilm formation and preformed Af biofilm, and Pa biofilm supernatants were more inhibitory than planktonic supernatants in both cases (P<0.001 for both comparisons). For comparison, a second analysis examined only the studies done in the presence of serum, and with all 16 non-CF isolates. For this second analysis we first compared the controls from each experiment where Pa isolates had been studied (in which only one type of Pa supernatant was used per experiment) by one-way ANOVA to assure the Af control results were not different in those experiments. These analyses showed that there were no significant differences in the Af controls. With this assurance we then compared the inhibition results of the three types of Pa for the two types of Pa supernatants. This allowed 20 experiments with the non-CF Pa’s and 10 experiments each with mucoid and non-mucoid CF Pa to be studied. The conclusions were the same as the prior statistical analysis, that of the studies done in the absence of serum.All of the studies done with or without serum appeared to indicate that there were differences in inhibitory activity between Pa planktonic and biofilm supernatants. Because there were few experiments, at this point, where these two types of Pa filtrates can also be suggested by visual comparison of Figs E. coli), there were direct comparisons of the two kinds of Pa supernatants, and supernatants from planktonically grown Pa were never more inhibitory than supernatants from biofilm-grown Pa, regardless of the source of the Pa.These statistical differences in planktonic and biofilm All the preceding studies assessed metabolic effects on biofilm. We now assessed effects on the physical biofilm itself.Pa spent culture filtrates on Af biofilm thickness and morphology were assessed using CLSM. The thickness results for the Af biofilm after conidia exposure for 16h to the Pa spent supernatants from the representatives of all three bacterial phenotypes are shown . Treatment with culture filtrates from the CF mucoid strain culture, grown under biofilm condition, resulted in decreased number of hyphae and presence of some "bulging" structures  distributed throughout the disperse filaments, and “glued” hyphae .Untreated biofilms showed an architecture formed by a dense filamentous multicellular structure with acute-angle dichotomous branching Figs  and 6A. ” hyphae . The CF preformed Af biofilm with the spent supernatants from the non-CF and the CF mucoid isolates were not associated with important morphology changes  present in the Pa biofilm culture was passed through an ultrafilter that now excluded all materials >30,000 MW, and tested against Af conidia forming biofilm, in comparison to the same filtrate not further manipulated (as in Af biofilm formation (P<0.001), and were not different from each other. We conclude that the inhibitory component(s) in Pa filtrates appear to be <30,000 MW species.To determine whether the inhibitors in filtrates were related to small molecular species, a sample of inhibitory filtrate from the non-mucoid ed as in . These tPa inhibiting Af, we grew E. coli ATCC 43888 under identical conditions as we had the Pa isolates, and collected these filtrates in the same manner. Supernatant from the non-mucoid Pa was included in these experiments as a positive control. The filtrates of the E. coli, either grown planktonically or as biofilm, did not significantly inhibit Af conidia forming biofilm .As a brief inquiry into the specificity of the culture filtrates of Af isolate, 10AF, was representative, three and nine other non-CF Af isolates were tested, by challenging formation of Af biofilm from their conidia, or preformed Af biofilm, respectively  by the addition of the Pa cells (data not shown).To ascertain whether the reference Af biofilm and that the inhibition by Pa is not unique to our reference strain of Af.We conclude that not all bacteria produce compounds that are inhibitory to Af biofilm by the Pa filtrates would be due to a reduced nutritional value of the spent culture filtrates diluting out the value of newly added medium during the incubation phase in the Af biofilm assays. The effect of fresh RPMI diluted with Pa culture filtrates (from biofilm-grown Pa) or sterile distilled water (a milieu that offers no nutrient support) on Af biofilm formation was studied. Fungal biofilm metabolic activity was inhibited over a range of ratios of fresh RPMI diluted with culture filtrates obtained from the representative CF and non CF Pa isolates, with inhibition starting at as little as 20% filtrate (80% fresh RPMI) (P<0.05)   Pa secretes into the culture filtrate, rather than nutrients removed by Pa.We addressed the possibility that the inhibition of (P<0.05) . In contd water) ; this cod water) . These fAf biofilm, it was of interest, for comparison, to see the effect of Pa supernatants on Af growing planktonically, as would occur in tubes, as in classical susceptibility testing [Pa groups was studied. When Pa supernatant was diluted in medium 1:1, exactly as in the challenges of biofilm, applied to Af conidia inocula in tubes, and the tubes examined when hyphal growth in matched controls was 4+, visible Af growth in the presence of supernatants did not occur . To again assure the inhibition seen was a result of toxic factors from the Pa, rather than depletion of media by Pa growth, the dilution series experiment with distilled water was repeated as above  reduced the planktonic growth metabolic activity by XTT virtually completely, whereas it was only when the concentration of distilled water reached 40% (only 60% fresh medium) that any effect at all was seen on that XTT result , using the 3 representative isolates detailed, and (a) in the absence of serum from all steps or (b) just during the generation of supernatants. Comparison was made to untreated filtrates.Two experiments were performed on heat treatment of the Pa isolates, under both test conditions “(a)” and “(b)”, removed all inhibitory activity against Af forming biofilm or preformed Af biofilm, i.e., heated supernatant results same as controls (data not shown). There was one small exception: the non-mucoid CF strain heated supernatant retained significant inhibitory activity against Af forming biofilm, but not against preformed Af biofilm, only when the filtrate was from the Pa biofilm (not planktonic Pa) culture, and only in condition “(b)”.The heat treatment of the supernatants of all 3 Af forming biofilm (both P<0.001 compared to untreated Af control), but this was significantly diminished compared to untreated supernatants (both P<0.001). When either frozen-thawed supernatant was tested against preformed Af biofilm, all activity was lost.In another experiment, freeze-thawed supernatant of the non-mucoid CF isolate, either grown planktonically or as biofilm, retained inhibitory activity against Af-inhibitory activity from supernatants of all types of Pa isolates. Freeze-thawing significantly diminishes inhibitory activity.We conclude that the heat treatment used removed essentially all Pa culture filtrate with DNAase or proteinase prior to assay for inhibitory activity. Compared to untreated Pa filtrate, filtrate treated with DNase I showed no significant differences in inhibitory capacity compared to Af control, nor did the DNase I alone have an inhibitory effect (data not shown).To determine whether the inhibitory substances were protein or DNA, we treated a nonmucoid Af biofilm compared to Af biofilm untreated controls (P<0.001). The EDTA inhibitory effect was less than that of untreated Pa filtrate. EDTA + DNase I did not affect inhibition of Af biofilm by Pa filtrate. Finally, DNase I had no effect on EDTA inhibition of Af biofilm, as there were no significant differences between EDTA and DNase I + EDTA.EDTA  alone reduced the metabolic activity of Pa filtrates did not affect filtrate inhibition of Af biofilm formation.Proteinase K alone did not affect Af biofilm formation. The addition of Proteinase K to Pa types tested, we also tested for some phenotypes previously associated with Pa virulence. Elastase activity, a virulence factor in Pa [Pa isolates that were used in the Af biofilm studies. The 5 non-mucoid CF isolates more frequently showed a positive test compared to the 5 mucoid CF and 5 non-CF isolates .Because of the differences we observed among the or in Pa , was assPa colonial types have been associated with lasR gene (a “master” gene controlling Pa virulence genes) mutations, more severe lung disease and persistence despite eradication attempts, including a wrinkly colony surface, irregular colony edge, metallic sheen, and green colony color [Af inhibition; 2 or 3 (of 5) of the non-mucoid isolates had each of these characteristics, compared to 1 to 3 of the other groups of 5 in each instance.Certain ny color . As withAf and Pa produce siderophores to acquire iron for their needs. Three experiments were performed to confirm a possible role for iron sequestration by Pa filtrates in the inhibition of Af. A filtrate of the inhibitory non-mucoid Pa, grown under biofilm conditions, was tested  . At 3000 μM FeCl3, there was no significant inhibition compared to control Af, and the inhibition was significantly less (P<0.001) than that with the filtrate without FeCl3. At 30 mM FeCl3 the XTT result showed not only lack of inhibition, but was significantly enhanced compared to both the filtrate-free control and filtrate without added FeCl3.Iron is a co-factor required for growth and both iltrate) . In comb3 alone, reported preliminarily elsewhere [3 alone at ≥2500 μM to enhance Af biofilm XTT activity. The enhancement of growth in the present study, with 3000 μM FeCl3 alone, compared to partial inhibition by the same concentration combined with filtrate, confirms the dose-responsive reversal of inhibition by filtrate shown in In separate studies on FeCllsewhere , we havePa supernatant inhibition, that iron sequestration, such as in the form of Pa siderophores or other Pa iron-binding molecules [Af biofilm by Pa filtrates.We conclude, from the iron reversal of olecules , is at lAf growth appears affected by any Pa isolate, and the inhibition is more intense than on Af biofilm growth, we show that the described inhibition of Af biofilm by Pa [Pa isolate. Titration studies may have elucidated differences, among Pa types, in inhibition by supernatants on planktonic Af growth, but we chose not to pursue this. The differential effects of Pa types on Af biofilm is consistent with the extensive phenotypic and genomic changes in the mutable Pa organism that, associated with chronic residence in CF airways, have been described [Pa cells inhibit the growth and formation of Af biofilm, with CF Pa isolates more inhibitory, and non-mucoid CF Pa isolates most inhibitory. Inhibition did not require the presence of Pa cells, as Pa culture filtrates were also inhibitory. The inhibition by filtrates showed the same hierarchy, with CF Pa isolates more inhibitory, and non-mucoid Pa isolates most inhibitory. Preformed Af biofilm was more resistant to Pa; non-mucoid CF Pa isolates were most inhibitory, and, again, that inhibition could be reproduced with culture filtrates. Reproducibility of the three Pa groupings was assured by the repeated experiments of various conditions, utilizing representative isolates of the groupings, as well as the number of isolates studied. Moreover, selection of Pa19, the most inhibitory of the non-CF isolates, as the representative of that group, would have minimized the differences in these studies between the CF and non-CF isolates.Whereas planktonic lm by Pa –45 is reescribed ,8,9,61. Af biofilm appears dependent on the bacterial growth conditions, since filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. However, the relationship of the number of Pa cells at the end of biofilm growth vs. the number after planktonic growth is unknown. Differences in biological effect on mammalian cells for molecules produced by planktonic Pa vs. biofilm Pa have previously been described, although planktonic Pa in that study produced more such active molecules [Af-inhibitory power by the three Pa groupings is unrelated to the growth of the Pa, based on our growth studies by representative isolates of each group. However, each of the groups studied may be heterogenous, and more extensive studies, with more members of each group, might then show a relationship between growth and supernatant inhibition.Inhibition of olecules . We haveAf inhibition by the XTT assay of metabolism (which does not exclude the possibility of death of some Af cells), because of the controversies in interpreting CFU reduction in a multinucleate filamentous organism, commonly with incomplete septa between cells [Af is, in fact, killed by Pa [Af biofilm show that Pa effects on Af are not solely on inhibition of the initial adhesion stage of Af biofilm development.We chose to measure en cells ,63. Suchen cells . Howevered by Pa . The XTTPa molecules that can explain the various inhibitions we have described [Pa-derived molecules, probably not nucleic acids or proteins per our studies, can explain at least most of the inhibition in supernatants. Whereas most previous work has focused on the toxic effects of Pa metabolites on fungi, our work suggests that denial of Fe to Af by Pa products is an important part of the inhibition. This is in contrast to a study of Pa inhibition of Candida albicans, which concluded that inhibition of that fungus was not a result of Fe limitation [Pa, as recent elegant work has shown that Af can transform Pa metabolites, and thus radically alter the effect on the interaction, including the degree of inhibition [Pa could also in part be a result of modulation of Af siderophore production by Pa metabolites [Previous work has indicated many candidate escribed –47. We cmitation , althoughibition . Finallyabolites .Pa quorum-sensing systems control expression of virulence factors, such as those previously implicated in Af inhibition [Pa isolates [Pa virulence [Af inhibition.hibition –47. A stisolates  might suirulence ,58 that Af biofilm was not inhibited by Pa [Pa isolates. Our findings also contrast with the report of heat-resistance of Af-inhibitory factors produced in Pa cultures [Pa and Af culture and/or heating conditions. These differences support the value of detailing the physical and chemical characterizations of the supernatants described here; these characterizations could lead to further identification of the molecules responsible for the inhibition we have described. Another pathway to this end would be to utilize the many Pa virulence factor mutants [Our findings contrast with others, who reported ed by Pa , a diffecultures , since w mutants  that havPa factors, whereas different regulatory systems may have different kinetics [Pa culture, and assess whether inhibitory factors are produced to a greater or lesser extent. We also cannot state at this time whether the enhanced inhibitory activity by biofilm Pa compared to planktonic Pa culture filtrates, or that resulting from non-mucoid CF isolates and their filtrates, is owing to increased production of inhibitory factors, or possibly the production of different factors. Nor is it known whether the inhibition by live cells relies solely on the same inhibitory factors (factors which can be released by Pa cells in close proximity to Af without dilution in culture medium) as in supernatants or whether there are unique factors resulting from or triggered by microbial contact. In this regard, the apparent greater inhibitory activity of live cells compared to supernatants .A limitation of our studies is that we have only studied one time period, although one adequate for bacterial growth, for production of kinetics . It may ts e.g.,  could bemicrobes . It woulelopment , since tisolates ; moreove setting . Thus, wPa isolates of varying colonial phenotype can co-exist in the same patient, but isolates with varying Af-inhibitory properties can co-exist; such polymorphism could be a transition state in the evolution of Pa colonization. Overall, from our studies of different Pa phenotypes, we hypothesize the transition of Pa from non-mucoid to mucoid [Pa’s inhibitory effect on Af biofilm, and thus, helps explain why establishment of Af in the airways usually occurs later in CF disease. Evolution of Pa may act in concert with other factors favoring Af colonization [Individual patient samples in our cohort shows not only that o mucoid –75, a prnization , includiS1 Table(DOCX)Click here for additional data file.


The results show that the cooling power per square meter collector and ice production per square meter collector per day depend largely on generation temperature and condensation temperature and they increase firstly and then decrease with increasing generation temperature. For every working fluid there is an optimal generation temperature at which organic Rankine efficiency achieves the maximum value. The cooling power per square meter collector and ice production per square meter collector per day are, respectively, 126.44 W m Li et al. [2 was employed to produce ice of about 4-5 kg day−1. Li and Wang [In recent years, there is an increasing need for cooling due to global warming, so, the energy consumption used for cooling has increased drastically , 2. The i et al.  developei et al.  presenteand Wang  presenteand Wang  proposedand Wang  presenteand Wang  studied and Wang  developeCurrently, the use of thermal energy to operate an ORC/VCC for air-conditioning and cooling has become the subject of renewed interest and has been reported by several investigators . AphornrIn a typical ORC/VCC, the maximum temperature at the inlet to the expander is limited to the temperature of the fluid leaving the solar collector. Because of the relatively low temperature capability associated with the flat plate collectors the coefficient of performance (COP) of an ORC/VCC is generally low. In this paper, to improve the COP of an ORC/VCC for ice making driven by solar energy, the parabolic trough collectors with higher temperature are selected to provide energy to heat and vaporize a working fluid, and a stationary modeling is developed in order to demonstrate the feasibility of such a cooling process.The system of ORC/VCC for ice making driven by solar energy mainly consists of solar collector (generator), expander, compressor, condenser for cooling system, throttle valve, evaporator for ice maker, condenser for power system, and working fluid pump, as shown in friction and heat losses in ORC/VCC are negligible;the power consumed by condensers for both ORC and VCC is negligible.To develop the thermodynamic model, the following assumptions are made:For ORC,For VCC,The overall COP of RC/VCC is defined asTboi is in the range of 60–160°C, the condensation temperature is 35–45°C, and the evaporation temperature is −5°C and keeps being invariable. For evaluating the feasibility of ORC/VCC for ice maker, the intensity of solar direct radiation is 300 W m−2 and the total solar direct radiation is 7560 kJ m−2 per day. The isentropic efficiencies for expander, compressor, and working fluid pump are, respectively, 0.85, 0.8, and 0.9, as shown in Considering the instability of solar heat source, the generation temperature s and ηt as a function of Tboi. In s and ηt depend largely on Tboi and they increase firstly and then decrease with increasing Tboi, as shown in Tboi on Pc and N are illustrated in s, ηt, Pc, and N have the same change trend with Tboi. For solar collector, the thermal efficiency, ηsolar, depends on both the solar radiation and the temperature difference between the generation temperature and ambient. When solar radiation, condensation temperature, and ambient temperature keep being invariable, ηsolar only depends on the generation temperature which can be controlled by changing the mass flow rate of working fluid for ORC. That is to say, ηsolar depends on the mass flow rate of working fluid for ORC. According to ,,s and ηectively . Pc is,Tboi can automatically be calculated by collecting solar radiation, condensation temperature, and evaporation temperature and then the mass flow rate of working fluid for ORC can automatically be adjusted by the frequency converter, working fluid pump, and programmable logic controller so that the solar ice maker is in the optimal operation state.The programmable logic controller and frequency converter are suggested to be adopted in practical use and the optimal s, ηt, Pc, and N are shown in Figures s, ηt, Pc, and N decrease with the increase of Tc. COPs is 76.40%, 64.09%, and 54.21%, ηt is 48.55%, 40.72%, and 34.45%, Pc is 150.49, 126.44, and 107.13 W m−2, and N is 9.06, 7.61, and 6.45 kg m−2 day−1 when Tc is 35, 40, and 45°C, respectively. Pc at condensation temperature of 35°C is 1.19 times larger than that at 40°C and is 1.40 times larger than that at 45°C. Observing the profiles from Figures s, ηt, Pc, and N.The condensation temperature varies with ambient and the effects of condensation temperature on COPN for air cooled condenser. However, the power consumption and system investment for air cooled condenser are also lower than those for water cooled condenser which needs to be equipped with cooling water pump or cooling tower. Thus the system performance and payback period should be comprehensively considered so as to decide which type of condenser should be used during practical design.The condensation temperature for air cooled condenser is about 45°C and for water cooled condenser is in the range of 35–40°C; using seawater especially as cooling medium the condensation temperature is about 35°C. Generally speaking, the condensation temperature for air cooled condenser is higher than that for water cooled condenser, resulting in the lower ηp and COPc are generally different when using different working fluids, leading to different COPs, as shown in c is, respectively, 3.63, 3.30, and 3.87 for working fluids of R600, R600A, and R245fa. As evident in Tboi has an important influence on ηp, and ηp increases with Tboi when Tboi < 120°C. When Tboi < 110°C, ηp for working fluid R600A is superior to those for two other working fluids; however, R245fa has the maximum ηp when Tboi > 110°C. Observing the profiles from ηp achieves the maximum values at Tboi = 130, 120, and 150°C for working fluids of R600, R600A, and R245fa, respectively; that is to say, the ice making system employing working fluids of R600, R600A, and R245fa will not achieve the optimal performance when Tboi is higher than the temperature corresponding to the maximum ηp.The system performance varies with different working fluids due to having different physical properties. To sum up, using organic Rankine cycle-vapor compression cycle for ice making driven by solar energy is feasible and the keys are to develop the expander and compressor with high efficiency, especially the compressor with high pressure ratio.The cooling power per square meter collector and ice production per square meter collector per day are, respectively, 126.44 W and 7.61 kg at the generation temperature of 140°C for working fluid of R245fa, which demonstrates the feasibility of solar powered organic Rankine-vapor compression ice maker.s, ηt, Pc, and N depend largely on Tboi and they increase firstly and then decrease with increasing Tboi, indicating that there is always an optimal Tboi at which Pc and N can achieve the maximum values, while Tboi can be controlled by adjusting the mass flow rate of working fluid for ORC.COPs, ηt, Pc, and N. Pc at condensation temperature of 35°C is 1.19 times larger than that at 40°C and is 1.40 times larger than that at 45°C, indicating that the ice making system equipped with air cooled condenser or water cooled condenser has different performances.The condensation temperature has an important influence on COPTboi at which ηp achieves the maximum values and the ice making system will not achieve the optimal performance when Tboi is higher than the optimal Tboi.The system performance varies with different working fluids due to having different physical properties. For every working fluid there is an optimal Solar powered ORC/VCC for ice making is researched and the effects of working fluid types, generation temperature, and condensation temperature on the system performance are analyzed by the development of a thermodynamic model, the following conclusions can be drawn.


Cymbidium has known antibacterial and antiedema activity and has been used as an ingredient in cosmetics and fragrances. The effects of Cymbidium ethanol extract (CYM) on allergic response and the underlying mechanisms of action have not been reported. Therefore, the purpose of this study was to determine the effect of CYM on allergic responses. Topical application of CYM was effective against immunoglobulin E (IgE)/dinitrophenyl-conjugated bovine serum albumin- (DNP-BSA-) induced degranulation of RBL-2H3 cells and anaphylaxis in ICR mice. An allergic dermatitis-like mouse model was used to evaluate the therapeutic potential of CYM in vivo. Continuous application of 2,4-dinitrochlorobenzene (DNCB) not only induced dermatitis in ICR mice but also aggravated the skin lesioning. However, the application of CYM decreased skin lesion severity, scratching behavior, and IgE levels. In addition, CYM downregulated the expression of the proinflammatory cytokines interleukin- (IL-) 4, IL-13, and tumor necrosis factor- (TNF-) α. Studies of signal transduction pathways showed that CYM suppressed the phosphorylation of spleen tyrosine kinase (Syk), an upstream molecule. It also inhibited the phosphorylation of Akt, phospholipase C- (PLC-) γ, and mitogen-activated protein kinase kinase kinase (MEKK). These results indicate that CYM may be effective in preventing and reducing allergic response and may have therapeutic potential as an antiallergic agent in disorders such as atopic dermatitis. The α chains directly bind the IgE heavy chains while β and γ chains are involved in signal transduction [εR1 on mast cells [εR1 by β- and γ-receptor tyrosine-based activation motifs [γ, and mitogen-activated protein kinase kinase kinase (MEKK) as well as PLC-γ-mediated cleavage of phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) [An allergic response is a hypersensitive immune reaction to a diverse and complex combination of environmental and genetic factors –3. It ocεR1) , 10. Fcεsduction , 12. Thest cells –15. Whenn motifs . Phosphote (IP3) . IP3 incte (IP3) , 19. Degα. These cytokines exert proinflammatory actions associated with the allergic response [Following mast cell degranulation, various factors are released such as histamine, leukotrienes, prostaglandins, and other mediators that induce smooth muscle contraction, deposition of collagen and other tissues, and vasodilation , 21. In response , 23.Cymbidium belongs to the orchid family, which is distributed throughout Southeast Asia, China, Japan, and Northern Australia. Various research studies have demonstrated that phenanthrene and phenylpropanoid extracted from the roots of Cymbidium exert antibacterial effects against hay bacillus, pneumobacillus, and Trichophyton rubrum [ Cymbidium has shown anti-inflammatory effects in a mouse model of carrageenan-induced edema [ Cymbidium by chromatography, are used as ingredients in cosmetics and fragrances [ Cymbidium extracts also have antioxidant potential [ Cymbidium root extracts and their mechanisms of action have not been reported. Therefore, we investigated the effects of the Cymbidium ethanol extract (CYM) on allergic responses in vitro and in vivo.n rubrum . Cymbidied edema . Linalooagrances . Aromatiotential . The antC. eburneo-kanran was cultivated according to the agricultural practices and methods of the Korea Rural Development Administration and harvested in Eumseong (GPS: E 128°62′ N 36°56′). For extract preparation, 400 g of dried C. eburneo-kanran root was extracted thrice with 2 L of ethanol for 1 day. The resulting extracts were filtered through Whatman No. 1 paper, combined, and concentrated using a rotary evaporator  at 40°C. The acquired dried extracts were subsequently lyophilized and processed into powder.2 atmosphere. Trypsin-ethylenediaminetetraacetic acid (EDTA) solution (Sigma-Aldrich) was used to detach the cells, which were washed with phosphate-buffered saline .RBL-2H3 is a rat cell line and was acquired from the American Type Culture Collection . The cells were cultured in Eagle's Minimal Essential Medium  supplemented with 100 units/mL penicillin-streptomycin  and 10% (v/v) fetal bovine serum  at 37°C in a humidified 5% CO ad libitum. The Institutional Animal Care and Use Committee  at Yonsei University  approved the protocol for this study.Seven-week-old male ICR mice were obtained from Orient Bio  and housed in cages maintained in an environment with the temperature at 20−22°C and humidity of 40−50%. All the ICR mice were given access to standard laboratory chow and water5 cells/mL) were seeded into 24-well culture plates and incubated overnight in MEM containing 10% (v/v) FBS at 37°C. To sensitize RBL-2H3 cells, they were incubated with antidinitrophenyl (anti-DNP) IgE at 400 ng/mL. After 12 h, the sensitized RBL-2H3 cells were washed with Siraganian buffer (pH 7.2), containing 25 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 4 mM potassium chloride (KCl), 0.4 mM magnesium chloride (MgCl), 110 mM sodium chloride (NaCl), 0.1% BSA, 5.6 mM glucose, 1 mM calcium chloride (CaCl2), and 40 mM hydrochloric acid (HCl), and were then treated with CYM  in Siraganian buffer at 37°C for 30 min. To activate RBL-2H3 cells, dinitrophenyl-conjugated- (DNP-) BSA, 50 ng/mL was added at 37°C for 15 min. Cell supernatants (30 μL) were transferred to a 96-well culture plate and mixed with 1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide  in 0.1 M citrate buffer for 1 h. To terminate the reaction, 0.1 M carbonate buffer was added, and the absorbance of the reaction solution was measured at 405 nm using a microplate reader.RBL-2H3 cells (1 × 105 cells/mL) were seeded into 24-well plates and incubated in MEM containing 10% (v/v) FBS at 37°C for 12 h. Anti-DNP IgE (400 ng/mL) was added to the medium and incubated for 12 h. Then, the medium was replaced with serum-free MEM containing CYM (0 or 200 μg/mL), followed by the addition of DNP-BSA. RBL-2H3 cells were washed twice with cold PBS and lysed, and protein was extracted using the PRO-PREP protein extraction kit  with a protease inhibitor containing 1 mM sodium vanadate (Na3VO4) and 10 mM sodium fluoride (NaF). A Bradford assay kit  was used for the quantitative analysis of protein according to the manufacturer's instructions. The protein lysates were separated using 12.5% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane  using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad) for 40 min.RBL-2H3 cells  1/2, anti-Erk 1/2, anti-phospho-c-Jun N-terminal kinase (JNK), anti-JNK, anti-phospho-p38, and anti-p38, which were purchased from Cell Signaling Technology . After primary antibody incubation, the membrane was washed twice with TBS-T and incubated with the anti-rabbit IgG horseradish peroxidase- (HRP-) linked secondary antibody  for 2 h at room temperature. The signal detection was performed using the EZ-Western kit , and the images were acquired and analyzed using an ImageQuant LAS4000 .The PVDF membrane was blocked for 1 h with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T). The blocked membrane was incubated with primary antibody overnight at 4°C (1 : 2000). The primary antibodies used were anti-phospho-Syk, anti-Syk, anti-phospho-PLC-μg of DNP-BSA in 200 μL PBS containing 3% Evans Blue was injected into the tail vein. After 30 min, the mice were euthanized and their ears were excised and then incubated overnight with 500 μL of formamide at 60°C. The amount of dye taken up was determined by measuring the absorbance at 620 nm using a microplate reader.The anti-DNP IgE antibody was injected intradermally into each right ear of 7-week-old ICR mice. After 1 day, CYM or diphenhydramine (DPH) was orally administered to the mice and, 1 h later, 200 μL of 0.5% DNCB solution, and then the lesions on the dorsal skin were topically treated with 200 μL of CYM (10 mg/mL) every 2 days for a total of six times. Subsequently, the AD-like mice were euthanized. We also evaluated the progression of the severity of the dorsal skin dermatitis of the AD-like ICR mice during treatment with 0.5% DNCB or 0.5% DNCB and CYM. Five conditions were evaluated, erythema, pruritus/dry skin, erosion, lichenification, and edema/excoriation. These conditions were then scored as follows based on severity: none = 0, mild = 1, moderate = 2, and severe = 3, and the sum of the individual scores was considered the dermatitis score. To ensure the credibility of the severity test, a researcher who was not associated with the experiment and was blinded to the treatments, determined the score. In addition, to observe scratching behavior, a behavioral characteristic of AD-like mice, we video-recorded and analyzed their behavior for 60 min during treatment with 0.5% DNCB or 0.5% DNCB and CYM. The number of scratches was counted in the video recordings by a similarly blinded researcher who was not associated with the study. Lastly, we histopathologically analyzed the skin by enucleating the dorsal lesions from mice in all three groups ; these lesions were fixed in 4% paraformaldehyde solution and cryoprotected in 30% sucrose solution. The skin samples were fixed in a cryomold using Tissue-Tek optimum cutting temperature (OCT) compound . The cryoprotected tissue samples were sectioned and stained with hematoxylin and eosin (H&E), and their thickness was measured using histopathological analysis with light microscopy .These experiments were performed according to the method of Park et al.  with sli™ Deluxe  according to the manufacturer's instructions by measuring the standard and sample absorbance values at 450 nm using a microplate reader.The serum IgE levels were measured using mouse blood collected from the heart. The serum was analyzed using a Mouse IgE enzyme-linked immunosorbent assay (ELISA) MAX® 96 instrument . The following primers were used: mouse IL-4, sense 5′-GGTCTCAACCCCCAGCTAGT-3′ and antisense 5′-GCCGATGATCTCTCTCAAGTGA-3′; mouse IL-13, sense 5′-CCTGGCTCTTGCTTGCCTT-3′ and antisense 5′-GGTCTTGTGTGATGTTGCTCA-3′; mouse TNF-α, sense 5′-CTGTAGCCCACGTCGTAGC-3′ and antisense 5′-TTGAGATCCATGCCGTTG-3′; and mouse β-actin sense 5′-ATGCCATCCTGCGTCTGGACCTGGC-3′ and antisense 5′-AGCATTTGCGGTGCACGATGGAGGG-3′. The denaturation, annealing, and extension conditions and number of cycles run were 94°C for 60 s, 49°C for 45 s, and 72°C for 45 s for 35 cycles, respectively.Total RNA was isolated from tissue extracted from the dorsal lesions using Tri-reagent  according to the manufacturer's instructions. The concentration of total RNA was determined using a spectrophotometer . Total RNA was used as a template for cDNA synthesis, which was performed using a cDNA Synthesis kit . The real-time polymerase chain reaction (qPCR) analysis was carried out using SYBR Green I and a LightcyclerP values < 0.05, <0.001, and <0.0001 were considered statistically significant.The experimental results are expressed as the mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) was used for multiple comparisons followed by a Tukey-Kramer post hoc analysis.  in vivo. We locally sensitized the ears of mice by injecting them with IgE, followed by intravenous injection of DNP-BSA and Evans Blue solution. The CYM treatment group showed a suppressed local allergic response, which was similar to that observed after treatment with DPH, an antihistamine  of CYM, the inhibitory effect increased by 230% compared with IgE/DNP-BSA treatment (μg/mL (data not shown).IgE/DNP-BSA-induced RBL-2H3 cell degranulation was measured using a reatment . In addiεR1 signaling. The phosphorylation of Syk was decreased by CYM treatment by about 2-fold compared to that observed after IgE/DNP-BSA treatment. The PLC-γ-induced cleavage of phospholipids is related to calcium influx. The phosphorylation of PLC-γ was strongly decreased by about 3-fold by CYM treatment group compared to that observed after IgE/DNP-BSA treatment. CYM treatment also suppressed the phosphorylation of Akt by about 2.5-fold compared with IgE/DNP-BSA treatment. The Erk 1/2, JNK, and p38 proteins, which are subfamilies of MAPK, and their phosphorylation were suppressed by about 3-, 2.7-, and 2-fold, respectively, by CYM treatment /AKT and the MAPK pathways [γ, PIP2, DAG, and IP3 occurs following calcium ion influx into the cytosol through calcium channels [ IL-4, IL-13, and TNF-α. CYM was effective in inhibiting the degranulation of RBL-2H3 cells by suppressing the phosphorylation of various proteins such as Syk and PLC-γ and that of proinflammatory cytokines through the inhibition of phosphorylation of Akt, Erk 1/2, JNK, and p38 (The initiation of an allergic response is followed by associated symptoms such as AD, anaphylactic shock, asthma, and conjunctivitis , 5. CYM se assay . The degpathways . Phosphochannels . An incr and p38 . Therefo and p38 .In conclusion, CYM had an antiallergic effect on dermatitis by inhibiting Syk-mediated signal transduction, suppressing proinflammatory cytokine gene expression and reducing serum IgE levels. We suggest that CYM may have therapeutic potential as an antiallergic agent for treating disorders such as AD.


CLEC‐2) and the collagen receptor glycoprotein (GP)VI activate platelets through Src and Syk tyrosine kinases, and phospholipase Cγ2. The initial events in the two signaling cascades, however, are distinct, and there are quantitative differences in the roles of proteins downstream of Syk activation. The activation of Akt and mitogen‐activated protein kinases (MAPKs) has been shown to enhance platelet activation by GPVI, but their role in CLEC‐2 signaling is not known.The C‐type lectin‐like receptor 2  MAPKs, but with a delay relative to Syk. Phosphorylation of these proteins was markedly inhibited in the combined presence of apyrase and indomethacin, consistent with the reported feedback action of ADP and thromboxane A2 in CLEC‐2 signaling. Phosphorylation of Akt and phosphorylation of ERK were blocked by the phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin and the protein kinase C (PKC) inhibitor Ro31‐8220, respectively, whereas Syk phosphorylation was not altered. On the other hand, both inhibitors reduced phosphorylation of the Akt substrate glycogen synthase kinase 3α/β (GSK3α/β). Phosphorylation of GSK3α/β was also blocked by the Akt inhibitor MK2206, and reduced at late, but not early, times by the MEK inhibitor PD0325901. MK2206 and PD0325901 inhibited aggregation and secretion in response to a low concentration of rhodocytin, which was restored by GSK3α/β inhibitors.The CLEC‐2 regulates Akt and MAPK downstream of PI3K and PKC, leading to phosphorylation and inhibition of GSK3α/β, and enhanced platelet aggregation and secretion.These results demonstrate that  C‐type lectin‐like receptor 2 (CLEC‐2) is a C‐type lectin‐like receptor that is highly expressed in platelets and expressed at a low level in other hematopoietic cells 2 (TxA2), and actin polymerization CLEC‐2 signals through a similar pathway to the collagen receptor gylcoprotein (GP)VI/FcRγ, which utilizes an immunoreceptor tyrosine‐based activation motif (ITAM) in the cytosolic tail of FcRγ The activation of Syk by CLEC‐2 or GPVI initiates a signaling cascade of adapter and effector proteins that culminates in the activation of phospholipase Cγ2 (PLCγ2) 2+/calmodulin‐dependent protein kinase kinase (CaMKK) independently of PI3K The phosphoinositide 3‐kinase (PI3K)–Akt pathway has been shown to support platelet activation by GPVI through the regulation of the serine/threonine kinase Akt  et al. Mitogen‐activated protein kinases (MAPKs) are divided into four subgroups: extracellular signal‐related kinases (ERK), c‐Jun N‐terminal kinases (JNK), big mitogen‐activated protein kinase 1 , and p38. Among these, ERK, JNK and p38 are expressed in platelets, and are regulated by a wide range of receptors. ERK is activated in response to collagen, and the MEK inhibitor U0126 blocks activation by low concentrations of collagen In this study, we investigated the role of the PI3K–Akt and MAPK pathways in platelet activation by CLEC‐2 by using the pharmacologic inhibitors MK2206 and PD0325901, which block Akt and the MAPK‐activating protein MEK, respectively. We show that Akt and ERK support platelet aggregation in response to low concentrations of the CLEC‐2‐activating snake toxin rhodocytin through phosphorylation of GSK3α/β, thus preventing its inhibitory action. Moreover, the PKC inhibitor Ro31‐8220 also reduces GSK3α/β phosphorylation. These results demonstrate that CLEC‐2 regulates GSK3α/β phosphorylation downstream of Akt, ERK, and PKC, thus enhancing platelet activation.Calloselasma rhodostoma venom as previously described Rhodocytin was purified from m sodium citrate, 110 mm glucose, and 80 mm citric acid), and centrifuged at 200 × g to obtain platelet‐rich plasma (PRP). Prostacyclin (0.5 μg mL−1) was added, and PRP was centrifuged at 1000 × g for 10 min to obtain a platelet pellet. The platelets were washed once by resuspension in HEPES–Tyrode's buffer  and further centrifugation at 1000 × g for 10 min in the presence of prostacyclin (0.5 μg mL−1) and 1 : 9 (v/v) acid citrate dextrose. The pellet of washed platelets was resuspended in a small volume of the HEPES–Tyrode's buffer, and then diluted to an appropriate concentration for experimentation: a cell density of 2 × 108 mL−1 was used for aggregation, and a cell density of 5 × 108 mL−1 was used for western blotting.All donors gave informed consent, and the study was approved by the University of Birmingham ethical review committee. Platelet preparation was performed as previously described m integrilin (eptifibatide), unless otherwise mentioned. Samples of washed platelets (300 μL) were stimulated with rhodocytin in an aggregometer at 1200 r.p.m. and 37 °C. Platelets were pretreated for 15 min with the following inhibitors : apyrase (2 U mL−1), indomethacin (10 μm), PRT‐318 (5 μm), PP2 (10 μm), BAPTA‐AM (10 μm), wortmannin (100 nm), Ro31‐8220 (5 μm), LY294002 (5 μm), MK2206 (1 μm), and PD0325901 (5 μm). An equal concentration of dimethylsulfoxide (0.2%) was added to the controls. Reactions were terminated by addition of an equal volume of ice‐cold 2 × lysis buffer . The samples were diluted with an equal volume of 2 × sample buffer , separated by SDS‐PAGE (10%), and transferred to a poly(vinylidene difluoride) membrane. Western blotting was performed with the indicated antibodies. Densitometry of the bands was performed with image j software .To inhibit aggregation, washed platelets were pretreated with 9 μAggregation was monitored by light transmission with a Born lumi‐aggregometer . ATP secretion was measured with a luciferin/luciferase substrate/enzyme mix (Chronolume).anova followed by the Newman–Keuls test. A P‐value of < 0.05 defined significant differences between test groups.All experiments were performed three to five times, and data are shown as means ± standard errors of the mean. Statistical analysis was performed with one‐way IIbβ3 blocker integrilin, demonstrating that phosphorylation is independent of outside‐in signaling by the integrin .In the present study, we investigated the role of the PI3K–Akt and MAPK pathways in platelet activation by CLEC‐2. As shown in Fig. 2 play a critical feedback role in the regulation of CLEC‐2 signaling in human platelets m), whereas phosphorylation of p38 was only slightly inhibited , and very slightly delayed the onset of aggregation in response to an intermediate concentration (30 nm). There was no inhibition of aggregation or secretion in response to 300 nm rhodocytin , a slight delay in onset at an intermediate concentration (30 nm), and no effect at a higher concentration  was delayed relative to phosphorylation of Akt, ERK, and GSK3α/β, which occurred after 90 s  but not a high (10 μg mL−1) concentration of CRP was also partially reduced in the presence of MK2206 and PD0325901  in the presence of CHIR‐99021 or SB216763 , but observed inhibition of the response to collagen , as previously reported The above results are consistent with a model in which rhodocytin stimulates GSK3α/β phosphorylation at its negative regulatory site downstream of Akt and ERK, thereby inhibiting GSK3α/β activity. To investigate the consequence of loss of phosphorylation of GSK3α/β on platelet activation, we treated platelets with the GSK3α/β inhibitors CHIR‐99021 and SB216763. These are ATP competitive inhibitors that block GSK3 activation but not its phosphorylation. Consistent with the inhibitory action of GSK3α/β, both inhibitors caused a small potentiation of aggregation by rhodocytin . Furthermore, inhibition of rhodocytin‐induced platelet aggregation and secretion by MK2206 was reversed in the presence of CHIR‐99021 . This is consistent with the observation in Fig. S1A that activation of Akt and MAPK by CLEC‐2 is also integrin‐independent .To verify that GSK3α/β is regulated downstream of Akt and ERK in CLEC‐2 signaling independently of αIn this study, we have demonstrated that CLEC‐2 activates the PKC, PI3K–Akt and ERK pathways, leading to phosphorylation of GSK3α/β at its negative regulatory site, and loss of its inhibitory action, thereby enhancing platelet activation at low concentrations of rhodocytin. Akt regulates phosphorylation of GSK3α/β at 90 s and 300 s, whereas ERK only regulates phosphorylation at the latter time. This study therefore adds to the growing evidence that GSK3α/β mediates a weak, constitutive inhibitory signal that regulates platelet activation by threshold agonist concentrations, and that this is inhibited by PKC‐mediated, Akt‐mediated and MAPK‐mediated phosphorylation of GSK3α/β, with the effect of PKC being mediated in part through secretion of ADP.2 in platelet activation by collagen than in platelet activation by CRP, which may accentuate the effect of other mechanisms of platelet regulation. Alternatively, collagen may regulate GSK3α/β downstream of α2β1, which is not activated by CRP. A concern regarding pharmacologic inhibitors is that of off‐target effects. In consideration of this, we have shown that two structurally different inhibitors of GSK3α/β potentiate platelet aggregation, thereby strengthening the argument that GSK3α/β is a negative regulator of CLEC‐2 signaling but that it has a negligible effect on GPVI signaling.The contribution of GSK3α/β to platelet activation has been controversial, as it appears to vary between agonists and is dependent on experimental conditions. For example, in the present study we showed that inhibition of GSK3α/β had a minimal effect on platelet activation by CRP, whereas collagen‐mediated activation was significantly reduced, as previously reported The explanation for the differential time course of phosphorylation of GSK3α/β by rhodocytin, with Akt inducing significant phosphorylation at both 90 s and 300 s, and ERK inducing it at later times, is not known. However, it is noteworthy that, although not significant, a clear trend towards reduction in GSK3α/β phosphorylation was seen at 90 s in the presence of PD0325901, and this may explain why inhibition of ERK activation by PD0325901 also reduces platelet aggregation by CLEC‐2 stimulation. In line with these results, ERK has also been shown to phosphorylate GSK3α/β in other cells The molecular mechanism through which GSK3α/β regulates CLEC‐2‐mediated platelet activation could be through phosphorylation of its substrate, MAP‐tau. Inhibition of GSK3β leads to a reduction in MAP‐tau phosphorylation in thrombin‐simulated platelets To date, there have been opposing reports on the importance of MAPK in platelets. The p38 inhibitor SB203580 has been reported to inhibit platelet aggregation induced by low concentrations of U46619 and collagen, although it is unclear whether this is attributable to off‐target effects on cyclooxygenase 2 are essential for robust activation of CLEC‐2 2 regulate phosphorylation of Akt and ERK by CLEC‐2. In our previous study m rhodocytin, and found complete blockade of CLEC‐2 activation in the presence of apyrase and indomethacin. In the present study, we observed a weak but significant increase in phosphorylation of Syk and PLCγ2 in response to a 10‐fold higher concentration of rhodocytin (300 nm). This demonstrates that the initial weak activation of CLEC‐2 by the snake toxin is significantly enhanced by the feedback action of ADP and TxA2. Furthermore, we have shown that blocking the effect of secreted ADP by apyrase or of secretion by Ro31‐8220 has a pronounced effect on Akt phosphorylation at 300 s but not at 90 s, which indicates two pathways of regulation, one of which is mediated by secretion of ADP. As CLEC‐2 activation is markedly inhibited in the presence of apyrase and indomethacin, however, we cannot readily compare the importance of CLEC‐2 and ADP in the regulation of Akt and MAPK phosphorylation. However, it noteworthy that, whereas ADP and TxA2 are important for reinforcing CLEC‐2 activation, neither ADP nor TxA2 induces CLEC‐2 phosphorylation We have previously shown that, together, secondary mediators such as ADP and TxAIn conclusion, we have shown that the Akt and MAPK pathways play a critical role in platelet activation at threshold levels of CLEC‐2 activation through phosphorylation of GSK3α/β, thus blocking its inhibitory action. PKC also regulates GSK3α/β phosphorylation at early times, independently of Akt.A. J. Moroi designed and performed experiments, analyzed data, created the figures, and wrote the manuscript. S. P. Watson designed experiments, wrote the manuscript, and supervised the work.The authors state that they have no conflict of interest.Fig. S1. Akt and MAPKs are activated after CLEC‐2 stimulation downstream of Syk and Src activation.Fig. S2. The PI3K–Akt and MAPK pathways are activated after GPVI stimulation.Fig. S3. The effects of PI3K, PKC, Akt and MAPK inhibition on GPVI‐mediated platelet aggregation.Fig. S4. The effects of GSK3α/β inhibitors on CLEC‐2‐mediated platelet activation.Fig. S5. GSK3α/β has a minimal effect on GPVI‐mediated platelet activation.Click here for additional data file.


Sagan-Zaba II, a habitation site on the shore of Siberia’s Lake Baikal, contains a record of seal hunting that spans much of the Holocene, making it one of the longest histories of seal use in North Asia. Zooarchaeological analyses of the 16,000 Baikal seal remains from this well-dated site clearly show that sealing began here at least 9000 calendar years ago. The use of these animals at Sagan-Zaba appears to have peaked in the Middle Holocene, when foragers used the site as a spring hunting and processing location for yearling and juvenile seals taken on the lake ice. After 4800 years ago, seal use declined at the site, while the relative importance of ungulate hunting and fishing increased. Pastoralists began occupying Sagan-Zaba at some point during the Late Holocene, and these groups too utilized the lake’s seals. Domesticated animals are increasingly common after about 2000 years ago, a pattern seen elsewhere in the region, but spring and some summer hunting of seals was still occurring. This use of seals by prehistoric herders mirrors patterns of seal use among the region’s historic and modern groups. Overall, the data presented in the paper demonstrate that Lake Baikal witnessed thousands of years of human use of aquatic resources. People and seals have formed long-lasting relationships in many parts of the world. This is especially true in the north, where these animals are a major part of societies’ economies and overall well-being, both presently and in the distant past –14. Undenerpa or Baikal seal (Phoca or Pusa sibirica), for several hundred thousand years [th through the 20th century, when seal products were used in various regional industries [Lake Baikal, Siberia  carried out excavations from 2006–2008 at the Sagan-Zaba II habitation site on the western shore of the lake . All necForaging groups were first present in Lake Baikal region in the Late Pleistocene, perhaps starting around 70,000 years ago , 50. ThePusa hispida; [nerpa can grow up to 1.8 m long and weigh as much as 130 kg, with the males tending to be slightly larger than the females [One of the key reasons for the density of human settlement in the Baikal region compared to adjacent areas of Siberia is that the lake and its nearby rivers offer an abundance of aquatic food resources , includihispida; , 58–59). females .For Baikal seals, the ice regime is a crucial ecological factor, as it affects many aspects of their life history and behavior. Lake Baikal freezes over completely from January to mid-May, and seals during this period distribute themselves unevenly across the lake. Just prior to freeze-over, females concentrate mainly in the deep open waters. When these regions of the lake are finally ice-covered in January, the females form dens within the ice and snow that are used as birthing areas for their pups. Adult males and juveniles try to extend their open-water feeding period as much as possible just before ice formation, and concentrate in areas that are ice-free. After the lake is ice-covered, the adult males are mostly found on the ice over the deeper portions of the lake, while the juveniles are more common nearer the shoreline. During the ice-cover period, seals separate from each other, with the spacing between their breathing holes and dens typically being greater than 100 m. Birthing usually starts at the end of February and lasts through the beginning of April. Lactation lasts approximately 2.5 months, partially overlapping with the mating period . Female With rising temperatures in April, breathing holes increase in size and leads (fissures) in the ice become more prevalent. At this time, seals lose up to 30% of their weight, as they spend significant time basking on the ice and moulting. During the ice break-up period in spring, from the second half of May until the end of June, the animals continue moulting and form aggregations of different densities on the remnant floating ice patches. In the summer, the animals can be found everywhere in the deep parts of the lake, and generally avoid its shallower sections. They often form aggregations on isolated rocky portions of the shoreline and on islands, sometimes consisting of up to a few thousand individuals. In the fall, the seals start to migrate to the north and northeast part of the lake to take advantage of the early forming ice, which forms a safer resting platform than the shoreline. The seals also sometimes form large groups on this first thin ice, or when resorting to using the shoreline as they wait for the ice to form. By January, they disperse into the deep-water areas of Lake Baikal, preparing for winter [Remains of Baikal seals are often found among the faunal specimens recovered from habitation sites along the west and north shores of the lake, as well as at a few sites along the eastern shore. Some also have been found within the graves of Middle Holocene foragers, and in animal sacrifice features created by the region’s Late Holocene pastoralists . HoweverSagan-Zaba cove is a ~200 m long funnel-shaped valley framed by steep hills, with a 5 m wide beach of cobbles and pebbles, and a 150 m wide terrace eroded by several small channels Figs . The ter3 [Test excavations at Sagan-Zaba revealed that some archaeological materials were present throughout the sediments in the floor of the valley, but were most well stratified and best preserved in a small area along the northeast shoreline. Our research thus centers on materials recovered from two trenches at Sagan-Zaba, numbered 4b and 4c , excavat3 , 63–64. The sediments in trenches 4b and 4c contained 20 lithological strata primarily formed of talus, colluvium, and proluvial sediments , 48. WhiChronological assessment of the archaeological materials recovered from the two trenches is based on 89 AMS radiocarbon dates on bones of ungulates and Baikal seals, all generated by the Oxford Radiocarbon Accelerator Unit. Analysis of radiocarbon dates and chronological evaluation of the site are provided elsewhere . Here weThe AMS radiocarbon dating of Sagan-Zaba II (based on the ungulate dates) indicates that the cove was first occupied starting around 9020 cal. BP, followed by a series of episodes of site use spanning much of the rest of the Holocene. Faunal remains from the earliest periods of site use, namely layers VII and VI, date from 9020 to 8650 and 8160 to 7880 cal. BP, respectively. Each of these layers individually appears to represent less than 250 years of site use, based on the calculated ETIs, which are shown at the 68% confidence interval in Chronology of the materials found within sublayer IIIB represent a complicated case. The dates from the layer range from 4400 to 2000 cal. BP, and the layer’s faunal remains and artifacts indicate that this layer contained materials deposited by both Middle Holocene foragers and later pastoralist groups  for detaFaunal remains from Sagan-Zaba II were identified using an extensive comparative collection assembled by T. Nomokonova and R. Losey and curated in Irkutsk, Russian Federation. The bulk of the 74,000 specimens recovered from the site consisted of mammal bone fragments that could not be more specifically identified. The identified specimens numbered 19,669, and 82.8% of these  were from Baikal seals . Given tBos and Equus), and seals, by analytical unit. Only specimens identified to at least the family level are included. To explore how the abundance of seal remains changed over time and in relation to other categories of fauna, we use standard quantification measures such as NISP (Number of Identified Specimens), and MNI . In addTo explore the potential differential effects of taphonomic factors on the preservation of faunal remains at the site, we first visually evaluated the collection. The overall state of preservation was assessed as relatively good in all layers, with the exception of stratum IVA, where some the skeletal elements displayed grainy and irregular surfaces, likely indicating post-depositional erosion . This maThe earliest evidence for seal use at Sagan-Zaba II is found in layer VII, which dates to ~9000 cal. BP Tables  and 2. SThe dominant taxonomic groups at the site, namely seals, wild ungulates, domesticated ungulates, and fish show several shifts in relative abundances during the course of site occupation . The smaTwo methods were used to reconstruct age at death for the seals represented at Sagan-Zaba. These are analyses of the incremental dentine bands in thin-sectioned canines and epiphyseal fusion of long bones. Dentine incremental structures of pinnipeds are widely accepted as a reliable technique for age determinations with an average error of +/- 1 year , 70. WebPhoca genus by Storå [Phoca/Pusa genera share similar sequences of epiphyseal fusion [Phoca sibirica, which are, as mentioned, genetically and morphologically similar to the ringed seals analyzed by Storå. In. Assessment of epiphyseal fusion is a relative ageing technique that involves assigning long bone elements to general age categories. This method has been developed for members of the by Storå  and prevby Storå , 72. Thel fusion . We assuSeal age groups at the site reconstructed using both methods are compared in os penis). Remarkably, only two small bacula fragments identified among the 16,000 seal remains at the site, and both were from juveniles in the site’s Middle Holocene layers. This could indicate the nearly complete absence of male seal remains at the site, but we believe that this conclusion is likely incorrect. The overall seal assemblage is dominated by yearlings and juveniles, and seals of this age are not visually sexually dimorphic and thus would be extremely difficult for even experienced hunters to differentiate [Sex assessments are difficult, as Baikal seals exhibit only minor sexual dimorphism in their crania , and no rentiate . The verrentiate ), and morentiate  indicateAssessment of the seasonality of seal use is based on season of death estimations made on incremental dentine bands in 74 canines, following the method established by Weber et al.  for BaikWhen the seasonality data are viewed in conjunction with the age assessments and observations of modern seal behaviour , it appeJuveniles (1–3 year olds) were mainly hunted from May to June, with a few taken in summer. Seasonality of death for the young adults (4–7 year olds) falls mainly in the spring, just like that seen with the majority of the yearlings, and it is possible that these individuals were nursing females, which would accompany the yearlings during this period of their development. A few younger adults also were taken at the beginning of summer, and these individuals may have been taken on the ice following the period of moulting, which is generally completed by seals of all age categories by the beginning of June. Some seals, particularly juveniles, likely were taken during the open-water season (June to September), when the seals intensively feed. It is also the only time of the year in which the seals regularly bask along the lake’s rocky shorelines , and perOverall, the seal season of death data fairly clearly suggests that human occupation of Sagan-Zaba cove occurred mostly in spring with some use also occurring in summer and early fall. This data of course cannot exclude human use of the cove during other seasons. Notably, other seasonal indicators, such as the trace quantities of migratory bird remains  at the site, also support the most use of this location during the warmer periods of the year.Seal remains at Sagan-Zaba II were found in direct association with other archaeological materials such as portable artifacts and features, and some skeletal elements from all layers were modified, all providing clear indication that humans were responsible for their accumulation at the site. No evidence of living structures was found at the site, but many features were present in all site layers, each being either simple hearths, or concentrations of faunal remains, other artifacts such as ceramics, or unmodified stones. Some of the more significant features are described below.Seal skeletal elements from all layers displayed butchery marks and traces of burning. While in layers VII through IIIB the percentages of seal specimens with cut marks is relatively low, ranging from 0.2–0.8%, the Late Holocene layers show far higher relative numbers of specimens with cut marks: 2.7% and 5.2% in layers IIIA and II-I, respectively. The relatively low number of seal remains with cut marks is perhaps not surprising given the fact that yearlings and juveniles dominate the seal assemblage at the site. Historically, yearlings weigh no more than 31 kg by the fall, and juveniles anywhere from 34–58 kg . In otheIn the earliest site layers (VII and VIB-A), seal remains were mostly found near hearths and simple stone features consisting of small piles of colluvium. Other artifacts from layer VII consist of 86 stone and bone items including microblades, prismatic stone inserts for bone tools, burins, a scraper, an abrader, and a quartzite sinker. Among the few bone implements from the layer are fragments of a harpoon head : 4, a poMiddle Holocene layers VB-A and IVB-A are very distinct in terms of spatial patterning of seal remains when compared to other site layers. These layers contained multiple features consisting largely of dense clusters of seal bone from multiple individuals, with the elements being disarticulated, the exception being a few cases of articulated vertebrae . For exaNo bone cluster features were present in the site’s late Holocene layers. Layer IIIB contained 796 stone artifacts, including scrapers, arrowheads, borers, drills, a knife, and a prismatic blade insert, and 514 pottery fragments also were present. Osseous tools present were a bone needle case, antler point, harpoon fragment, and an arrowhead; a single bronze awl also was present in the layer. The Late Holocene artifacts from layers IIIA and II-1 contained many metal items such as simple iron awls, and a Chinese bronze coin and a finger ring also were recovered. Bone items from the layers consist of a spoon made from a seal scapula : 6, threSagan-Zaba cove provides ample evidence of seal hunting throughout much of the Holocene, and within this time span several trends are apparent. First, sealing began here at least 9000 years ago, which to our knowledge is the earliest clear evidence for such practices in North Asia. This Early Holocene onset of sealing at Sagan-Zaba is consistent with the previously existing evidence for early seal hunting at Lake Baikal, which consisted of only a handful of remains, also apparently dating to the Early Holocene , 40, 44.Second, the peak of seal use at the site is the Middle Holocene (layers V and IV), when seals overwhelming dominate the faunal assemblages (> 90% of the NISP) and were deposited at far higher rates than observed during any other period of site use. In this period the foragers occupying Sagan-Zaba appear to have been seasonally specialized seal hunters. This sealing in many ways mirrors that seen in the region during the historic period . Namely,The peak in sealing at Sagan-Zaba appears to have begun at ~6750 cal. BP, almost precisely at the same time as the broader region experienced a discontinuity in the use of formal cemeteries during the Middle Neolithic . This dPinus sylvestris; [Third, the other major shift in subsistence practices at the site occurs in the Late Holocene with the introduction of domesticated herd animals, which first appear in layer IIIB, or some time between 4440 and 2000 cal. BP. Remains of fish and wild ungulate also are relatively more common at the site following the Middle Holocene. Remains of domesticates are initially a very small component of the faunal assemblage, and only become relatively numerous some time after ~2000 cal. BP. This pattern closely follows that seen at the Bugul’deika II habitation site, located about 40 km southwest of Sagan-Zaba along the open shoreline of the lake. At Bugul’deika, the earliest directly dated remains of domesticates (a horse), are ~2800 cal. BP old, but only after ~2000 cal. BP are such animals abundant at the site –39. The vestris; ). A secovestris; . How thevestris; .14C dates substantially older than their real age [Importantly, the Late Holocene deposits at both Sagan-Zaba II and Bugul’deika II contain some remains of seals and fish, indicating that these pastoralist groups also procured and consumed these animals, which is entirely consistent with historical practices of herding groups from this region . Subsistreal age . The culreal age .Examination of Sagan-Zaba’s faunal assemblage, including its chronology, association with other artifacts, and relations to climate and environmental changes, have provided a series of insights on sealing practices on Siberia’s largest lake. Some seal hunting first occurred at this site at least 9000 years ago, and by the Middle Holocene was regularly practiced from Sagan-Zaba cove, when foragers focused their efforts on procuring young seals basking on the spring ice. Seals were butchered and consumed at the site during all periods of occupation, including by pastoralists groups, who first utilized the cove some time just prior to 2000 cal. BP. Fishing and hunting of terrestrial fauna, mostly ungulates, were relatively minor activities at Sagan-Zaba II during the Middle Holocene period, when the site was most frequently utilized—sealing clearly was the predominant subsistence activity during this period. Fishing was relatively more important during the earliest periods of site use, and again in the Late Holocene, the latter period also witnessing the increasing importance of ungulate game hunting when compared to the Middle Holocene.Future research on sealing at Lake Baikal will undoubtedly have to come to terms with how this record was affected by a series of increasingly well-documented climate shifts, episodes of human migration and demographic change, and fluctuations in sociopolitical and ideological systems. This paper is one important step in this direction. Finally, it is clear that humans’ adaptations to Lake Baikal aquatic resources have a temporal depth that rivals that of some of the world’s marine coastal regions, particularly in terms of the antiquity of intensive pinniped use . Lake Ba


Inhibitory neuronal activity is critical for the normal functioning of the brain, but is thought to go awry during neurological disorders such as epilepsy. Animal models have suggested both decreased and increased inhibition as possible initiators of epileptic activity, but it is not known if, or how, human inhibitory neurons shape seizures. Here, using large-scale recordings of neocortical single neurons in patients with secondarily generalized tonic-clonic seizures, we show that fast-spiking (FS) inhibitory activity first increases as a seizure spreads across the neocortex, impeding and altering the spatial flow of fast epileptic traveling waves. Unexpectedly, however, FS cells cease firing less than half-way through a seizure. We use biophysically-realistic computational models to show that this cessation is due to FS cells entering depolarization block as a result of extracellular potassium accumulation during the seizure and not because they are inhibited by other inhibitory subtypes. Strikingly, this absence of FS inhibitory activity is accompanied by dramatic increases in local seizure amplitude along with unobstructed traveling waves and is seen during all secondarily generalized seizures examined, independent of etiology or focus. FS cessation also leads to prominent spike-and-wave events, suggesting that FS cell dynamics control the transition between the tonic and clonic phases of these seizures. Thus, it may be possible to curtail human seizures by preventing inhibitory neurons from entering potassium-dependent depolarization block, a novel and potentially powerful therapeutic avenue in treating multiple kinds of epilepsies.