Metabolic inhibition induced by the protonophore FCCP  resulted in a dose-dependent reduction of LTCC current  which was more pronounced during β-adrenergic stimulation with isoprenaline. ICa,L was still reduced by metabolic inhibition even in the presence of 3 mM intracellular ATP, or when the cell was dialysed with cAMP or ATP-γ-S to induce irreversible thiophosphorylation of LTCCs, indicating that reduction in ICa,L is not due to ATP depletion and/or reduced phosphorylation of the channels. However, the effect of metabolic inhibition on ICa,L was strongly attenuated when the mitochondrial F1F0-ATP-synthase was blocked by oligomycin or when the cells were dialysed with the non-hydrolysable ATP analogue AMP-PCP. Moreover, increasing the intracellular pH buffering capacity or intracellular dialysis of the myocytes with an alkaline solution strongly attenuated the inhibitory effect of FCCP on ICa,L. Thus, our data demonstrate that metabolic inhibition leads to excessive ATP hydrolysis by the mitochondrial F1F0-ATP-synthase operating in the reverse mode and this results in intracellular acidosis causing the suppression of ICa,L. Limiting ATP break-down by F1F0-ATP-synthase and the consecutive development of intracellular acidosis might thus represent a potential therapeutic approach for maintaining a normal cardiac function during ischemia.Metabolic stress evoked by myocardial ischemia leads to impairment of cardiac excitation and contractility. We studied the mechanisms by which metabolic inhibition affects the activity of L-type Ca During myocardial ischemia, mitochondria undergo progressive damage, including dramatic decrease in the activity of oxidative-phosphorylation complexes -phenyl-hydrazone (FCCP) had no significant effect on basal ICa,L at 30 to 70 nM but reduced the current at 100 nM concentration by 22.1 ± 9.6% , the inhibitory effect of FCCP was more pronounced, and was already significant at 70 nM. At 100 nM concentration, FCCP suppressed the ISO-stimulated ICa,L by 70.4 ± 6.6%  , another uncoupler of oxidative phosphorylation, caused a similar reduction of ISO-stimulated ICa,L as 100 nM FCCP  , an inhibitor of respiratory chain complex IV, or antimycin A (30 nM), an inhibitor of complex III, decreased basal ICa,L by 46.6 ± 13.6%  and 71.9 ± 2.4% , and ISO-stimulated ICa,L by 66.3 ± 5.4%  and 62.5 ± 2.9% , respectively  . Applicationship  or inacttionship .To exami P<0.05) . Sodium ectively . These rCa,L due to insufficient intracellular ATP levels. Therefore, we evaluated the significance of the presence of ATP in the pipette solution for the maintenance of ICa,L. In control conditions, when cells were dialyzed with a solution containing 3 mM ATP, the mean basal ICa,L amplitude was 3.1 ± 0.3 pA/pF (n = 17). In the presence of ISO (1 μM), ICa,L increased ~7-fold to 20.0 ± 1.3 pA/pF (n = 18). When ATP was excluded from the internal solution  and increased only by ~4-fold to 7.3 ± 1.7 pA/pF in the presence of ISO (n = 4)  . Additio4)  had no effect on either basal (not shown) or ISO stimulated ICa,L  via the patch pipette, thus bypassing the activation β-adrenergic receptor and adenylyl cyclase. Dialysis of the cell with cAMP stimulated ICa,L to 21.5 ± 4.2 pA/pF (n = 4) and the amplitude of the current was not significantly different from that obtained with 1 μM ISO. Ca,L stimulated by cAMP, and the effect was not significantly different from the one observed when the current was stimulated by ISO. Thus, the suppression of ICa,L during metabolic inhibition does not involve a reduction in cAMP production due to lack of ATP, but rather involves mechanisms located downstream from β-adrenergic receptor and adenylyl cyclase. In the second series of experiments, to test whether metabolic inhibition induced reduction in ICa,L is determined by insufficient phosphorylation of LTCCs, the patch pipette solution containing 3mM ATP was supplemented with the hydrolysis-resistant analogue of ATP, adenosine 5′-O-(3-thiotriphosphate) , which leads to irreversible thiophosphorylation of proteins i has been shown to reduce the activity of LTCCs [2+]i changes in the effects of metabolic inhibitors on ICa,L, Mg2+ in the pipette solution was substituted with Na+. The absence of Mg2+ in the internal solution led to an increase in basal ICa,L (7.7 ± 1.2 pA/pF (n = 4) vs. 3.1 ± 0.4 pA/pF (n = 7) in control), but no significant effect on the ISO-stimulated ICa,L was observed. In addition, FCCP (100 nM) still inhibited ICa,L in Mg2+-free solution similarly to when Mg2+ (4 mM) was present in the patch pipette solution   and [Mg2of LTCCs –32. To tsolution . These rCa,L, myocytes were dialyzed with alkaline (pH 8.3) internal solution and the effect of FCCP on ICa,L was tested. Dialysis of myocytes with alkaline solution strongly attenuated the inhibitory effect of FCCP on ICa,L. As shown in Ca,L than 100 nM FCCP in cells dialysed with control solution with pH 7.3.Development of intracellular acidosis during metabolic inhibition in cardiomyocytes is well documented , 19, 20.Ca,L, the effect of FCCP was examined in cells dialysed with an internal solution with a low pH buffer concentration (1 mM HEPES instead of 10 mM in control conditions). As expected, reducing the pH buffering capacity in the pipette solution amplified the inhibitory effect of FCCP on ICa,L as evidenced by a shift to lower FCCP concentrations of the concentration-response curve for the effect of FCCP on ISO-stimulated ICa,L  The mean peak amplitude of basal ICa,L in control and during exposure of myocytes to the increasing FCCP concentration (n = 5). * P<0.05 vs. control.(A) Dose dependent effect of FCCP on the peak amplitude of I(PDF)Click here for additional data file.S2 Fig3 on the peak amplitude of basal and ISO-stimulated ICa,L. The current traces shown in the top panel were recorded at times indicated by the corresponding letters on the main graph.A typical experiment representing effect of NaN(PDF)Click here for additional data file.S3 FigA time course of changes in ICa,L peak amplitude during the application of increasing FCCP concentrations to the ventricular myocytes dialyzed with internal solution containing 3 mM ATP and supplemented with 3 mM of ATP-γ-S. A transient application of ISO was used to induce irreversible thiophosphorylation of proteins. The current traces shown in the top panel were recorded at times indicated by the corresponding letters on the main graph.(PDF)Click here for additional data file.S4 FigCa,L suppression in the presence of 30 μM of antimycin in a myocyte dialyzed with a pipette solution in which ATP was substituted with non-hydrolysable ATP analogues AMP-PCP (3 mM) and ATP-γ-S (0.5 mM). The current traces shown in the top panel were recorded at times indicated by the corresponding letters on the main graph.An experiment demonstrating the lack of I(PDF)Click here for additional data file.


Interest in brines in extreme and cold environments has recently increased after they have been found on Mars. Those brines can be potential new subsurface habitats for peculiar ecosystems. In the McMurdo Dry Valleys of the Antarctic, the best analogue for Mars conditions, only a few cases of brines have been identified in some perennially frozen lakes and in one case in an underground aquifer. Here, we present the occurrence of pressurized brines in a shallow perennially ice-covered lake south of 70°S in an ice-free area of Victoria Land, Antarctica. For the first time, we also imaged, by means of ground penetrating radar data, the existence of a pingo-like-feature (PLF) formed by the extrusion of brines, which has also been confirmed by borehole evidence. Those brines are fed by an underground talik external to the lake basin, enhancing the possibility of unexploited ecosystems that could find an analogue in Martian environments. Interest in brines in extreme and cold environments has increased after they have been found on Mars13347810713et al.Lake sediments are potential palaeoclimate archives, and in Antarctica they have mainly been studied above 70°S, where the onset of the sedimentation is generally recorded to have occurred after the Last Glacial Maximum (LGM), as documented in maritime Antarctica between 28 and 21.1 kyr15171819South of 70°S, fresh water lakes are extremely rare and are mainly concentrated in coastal areas such as the case of Amery Oasis, where sedimentation started at approximately 12 cal kyr BP, and a core documented clear warming event at approximately 8.5 cal kyr BPAmong perennially ice covered lakes, only the large lakes of the McMurdo Dry Valleys have been extensively studied. In the Dry Valleys during the mid to late Holocene, relevant drawdowns and even complete desiccation events have been recorded, for example in the Taylor Valley2325The paper aims to I) show the first observed case of a small perennially frozen lake south of 70°S (74°S) in which brines were identified through ground penetrating radar (GPR) investigations and sampled by successive coring, II) present the first case of a pingo-like-feature (PLF) formed by brines coming from an open talik system, and III) contribute to the understanding of the palaeoclimatic significance of the lacustrine sediments found in the lake that started to accumulate not prior to 12.4 cal kyr BP. The first two aims could contribute to a better understanding of the possible analogies between this part of continental Antarctica and the Martian surface.The Tarn Flat area is the largest ice-free area (ca. 11 × 9 km) north to the McMurdo Dry Valley in Victoria Land (Antarctica) and is not far from the Italian Antarctic Research Station  of approximately −14 °C (considering the last 20 years) while precipitation, which is always in the form of snow, ranges between 100 and 200 mm/yr30The study area presents a very large number of lakes and ponds (64) that have dimensions from a few tens to several hundreds of metres . Generalet al.Guglielmin 2. The frost mound is located in the central part of the lake  profiles using combined 500 MHz and 1.6 GHz antennas , with a Lake-1 is scoured in granitic bedrock. It is not symmetric and has two troughs, one of which is deeper, has a bedrock depth that exceeds 7 m  and 4, iOnly in correspondence with the deeper trough is a relevant structure clearly visible that has lateral slopes, which were imaged by GPR  that correspond to the sediment layers found in the borehole . In detaThe thicker sediment layers H3 and H4 found in the borehole between 4.94 and 5.68 m were not imaged by the GPR just below the frost mound due to the extremely high attenuation of the electromagnetic waves that was most likely produced by the brines. In any event, such layers were interpreted on all the GPR profiles down to a depth of approximately 5 m below the lake surface  and 4.Moreover, the morphology of the sedimentary basal level (H4), which is characterized by a marked deepening as obtained by the 3D data integration shown in The 3D reconstruction obtained by integrating the top of the bedrock (BD) and the top of the sedimentary layer H3 is provided in Based on a visual observation of the amount and distribution patterns of the included bubbles and/or sediment, the borehole presents different types of ice. The two more common ice types can be summarized as type I, which contains a certain amount of fine sediments (clay/silt) that are randomly distributed and bubbles that are in the main not elongated, and type II, which is constituted by alternating ice I and pure ice with primarily horizontally elongated bubbles.The borehole stratigraphy  is quiteAt different depths, thin (1–2 cm thick) layers of sediments are present; they are silty-sand at the surface, at 1.51 and at 1.86 m H0 in  and siltBetween depths of 3.61 m and 3.78 m, there is a 17 cm layer of ice with bubbles that is elongated perpendicular to the surface, followed by a pocket of liquid brine (B1) with an important flow until a depth of 3.98 m, where a 12 cm layer of ice with some organic material inclusions occurs. The thickest (0.84 m) pocket of liquid and flowing brines (B2) lies below.Finally, between 4.94 m and the bottom of the borehole (5.68 m), there are two layers of frozen sediments  in an open system. A partial validation of such a hypothesis is the sensible pressure observed in the borehole, where an uplift of brine level B2 was observed with a flow rate that decreased with time (average of 9.6 l/hour). Such brines reached the lake surface approximately 40 hours after the borehole was drilled. It is certain that the pressure was much stronger in the past because remnants of brine with fragments of silty-sandy layers of sediments and algae are actually on the surface of the mound. Moreover, the fact that the salinity of the deeper brines (B2) is lower than that of the upper ones supports the hypothesis of a hydraulic open system because recharge from outside the lake basin can decrease the salinity that should otherwise increase in a closed system for repeated basal freezing events.The only pressurized brine system confined beneath ice and sediment layers similar to the case herein described has been recently found and reported at Lake Vidaet al.et al.The extension of B1 and B2 is limited within the deeper trough  within the ice both in the PLF and in the rest of the lake could have a similar origin to those found in Lake Vida, according to Dugan The deeper sediment layers H3 and H4 suggest a different origin, as proposed in 28et al.Recently, Anderson Considering that there is no evidence for higher levels of Lake-1, this small basin could preserve at least all the Holocenic hydrological and climatic changes although within a more limited thickness, for instance Lake Vida, where 8600 years are recorded in 27 m of ice and sediments; in other words, the sedimentary sequence of Lake-1 could be a more condensed chronological series.Regarding the age of the PLF and therefore the age of the possible brine flows, we can only hypothesize that this event postdates the sedimentation of layer H0 because all the sediment layers within the ice are truncated by the slope of the PLF, but we cannot exclude that this feature was formed by successive episodes that cut the different sediment layers at different times.We used a ProEx  GPR produced by Malå Geosciences that was triggered by an electromechanic odometer and equipped with two shielded antenna pairs with central frequencies equal to 500 and 1600 MHz. In the present work, we will only show examples taken from the 500 MHz dataset. A Trimble GPS that recorded a fix every 10 seconds was used for the absolute data positioning. The mean trace interval equalled 15 cm.14C dating. During the coring, when we reached the first pocket of brine at 378 cm we interrupted the coring and collected brine in aquifer B1 using a peristaltic pump and sterile tubing. After collecting 5 litres of brine B1, we continued the core sampling of the following 12 cm and then re-inserted a new sterile tube to collect the brine from aquifer B2, which was located between 410 cm and 494 cm of depth. Finally, after collecting brine B2, we completed the core, reaching a depth of 568 cm.In addition, a 51 mm diameter borehole that reached a depth of 5.68 m was drilled in the centre of the frost mound using a semi-portable core auger  and 2, eWe applied a processing flow to GPR data that included drift removal (zero-time correction), background removal, geometrical spreading correction and exponential recovery, bandpass filtering, 2D migration , and depth conversion. For the last two steps, we used a constant velocity value of 0.17 m/ns, considering both the reference value for pure ice and the results of dedicated diffraction hyperbola analyses, which all produced values very close to the previous one.For a better and more constrained GPR data interpretation, we calculated GPR attributes that were then integrated and correlated . Such a 42The salinities and pHs of the brines were measured at MZS and at Messina CNR laboratories using refractive index and a potentiometric method, respectively.14C dating samples were sealed in polyethylene bags and frozen at −20 °C. They were kept frozen until they were sent to the Beta Analytic laboratories  for radiocarbon dating. There the frozen sediments were subjected to acid washes following Beta Analytic standard procedures. After pre-treatment, the samples for radiocarbon dating were prepared for AMS by converting them into graphite. The calibrated ages were calculated with the OxCal 4.2 software program14C yr BP) ± σ and as calibrated age ranges with a 2σ error (95.4%) .The How to cite this article: Forte, E. et al. Pressurized brines in continental Antarctica as a possible analogue of Mars. Sci. Rep.6, 33158; doi: 10.1038/srep33158 (2016).


E. coli, and purified by the combination of affinity chromatography and gel filtration. The MSTN-inhibitory capacities of these proteins were examined in an in vitro gene reporter assay. A MBP-fused, truncated MSTNpro containing residues 42–175 (MBP-Pro42-175) exhibited the same MSTN-inhibitory potency as the full sequence MSTNpro. Truncated MSTNpro proteins containing either residues 42–115 (MBP-Pro42-115) or 42–98 (MBP-Pro42-98) also exhibited MSTN-inhibitory capacity even though the potencies were significantly lower than that of full sequence MSTNpro. In pull-down assays, MBP-Pro42-175, MBP-Pro42-115, and MBP-Pro42-98 demonstrated their binding to MSTN. MBP was removed from the truncated MSTNpro proteins by incubation with factor Xa to examine the potential role of MBP on MSTN-inhibitory capacity of those proteins. Removal of MBP from MBP-Pro42-175 and MBP-Pro42-98 resulted in 20-fold decrease in MSTN-inhibitory capacity of Pro42-175 and abolition of MSTN-inhibitory capacity of Pro42-98, indicating that MBP as fusion partner enhanced the MSTN-inhibitory capacity of those truncated MSTNpro proteins. In summary, this study shows that MBP is a very useful fusion partner in enhancing MSTN-inhibitory potency of truncated forms of MSTNpro proteins, and MBP-fused pig MSTNpro consisting of amino acid residues 42–175 is sufficient to maintain the full MSTN-inhibitory capacity.Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth. MSTN propeptide (MSTNpro) inhibits MSTN binding to its receptor through complex formation with MSTN, implying that MSTNpro can be a useful agent to improve skeletal muscle growth in meat-producing animals. Four different truncated forms of pig MSTNpro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in  Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily and negatively regulates skeletal muscle growth and development with little effect on other tissues –3. SimilActivation of MSTN from the latent state involves cleavage of MSTNpro by members of the bone morphogenetic proteins-1/tolloid (BMP-1/TLD) of metalloproteinases , 15. TheAccording to a study , a regioE. coli [E. coli expression system [In a previous study, we have successfully produced bioactive metalloproteinase-resistant mutant form of pig MSTNpro in E. coli  in an efn system , the detpMAL-c5x expression vector , PCR was performed using Q5 High-Fidelity DNA polymerase  with overlapping primer sets . After an overnight growth at 37°C, the pMAL-c5x-Pro42-218, pMAL-c5x-Pro42-175, pMAL-c5x-Pro42-115, and pMAL-c5x-Pro42-98 plasmids were extracted to confirm correct insertion by colony PCR across the assembly junctions or by DNA sequence analysis. E. coli strain of K12TB1  was transformed with the expression constructs by the heat-shock method, and spread on Luria-Bertani (LB)  agar plates containing 50 μg/mL ampicillin.Construction of plasmid containing full cDNA sequence of pig MSTNpro with mutation at the BMP-1/TLD proteolytic site (GenBank Accession No. KT932616) was reported previously , and thimer sets . The PCR°C overnight, and the 5 mL cultures were used to inoculate 250 mL LB media containing 50 μg/mL ampicillin. Protein expression was induced by adding IPTG to a final concentration of 0.3 mM when the cell density reached to an OD600 of 0.4 ~ 0.5, then the cultures were grown at 25°C for 15 hr. Cell pellets were collected, and soluble fractions of cell lysates were prepared, and maltose binding protein (MBP)-fused pig MSTNpro (MBP-MSTNpro) proteins were affinity-purified from soluble cell lysates using amylose resin as described previously [As described in detail previously , selecteeviously . After SThe pooled, affinity-purified MBP-MSTNpro proteins were applied to a Superdex 75 column  equilibrated with amylose affinity column buffer and eluted with the same buffer at a flow rate of 0.6 mL/min in a FPLC system. The absorbance was monitored at 280 nm, and 0.5 mL fractions were collected. The fractions were subjected to SDS-PAGE analysis to examine the purification.The ability of pig MSTNpro proteins to bind to MSTN was examined using a pull-down assay as described in detail previously . Three hSDS–PAGE was performed according to the method of Laemmli . SamplesAfter blocking with Tris-buffered saline  plus 0.01% Tween 20 (TTBS) for 2 hr at room temperature, the membranes were incubated with monoclonal anti-MSTN antibody  at a 1:1in vitro were measured using the pGL3-(CAGA)12-firefly luciferase reporter assay in HEK293 cells stably expressing (CAGA) 12-luciferase gene construct [2. 1 nM of MSTN (R&D Systems) or GDF11 (R&D Systems) plus various concentrations of commercial mouse MSTNpro (R&D Systems) or pig MSTNpro proteins in DMEM without serum were added to each well after removing the medium, and incubated for 24 hrLuminescence was measured using Veritas microplate luminometer  after adding Bright-Glo luminescence substrate . The % inhibition of MSTN activity was calculated by the following formula: % inhibition = (luminescence at 1 nM MSTN—luminescence at each ligand concentration)*100/(luminescence at 1 nM MSTN–luminescence at 0 nM MSTN). The MSTN-inhibitory activities were analyzed by regression analysis using Prism5 program . To examine the differences in MSTN-inhibitory capacity of these proteins and peptides, IC50 (ligand concentration inhibiting 50% of MSTN activity) values were estimated using a non-linear regression model defining dose response curve. The equation for the model was as follows: Y = Bottom + (Top–Bottom)/(1 + 10^(X–LogIC50)), where Y is % inhibition, Bottom is the lowest value of % inhibition, Top is the highest value of % inhibition, and X is Log ligand concentration. IC50 values were analyzed by ANOVA  using the same program.As described in detail previously , capacitonstruct . Cells  at 4°C overnight with 2 changes during dialysis. After dialysis, the proteins were incubated with factor Xa protease (New England BioLabs) for 24 hr at 4°C to remove MBP from these proteins. One ng of factor Xa was used to digest 50 ng of MBP-fusion proteins. The digestion was examined by SDS-PAGE. The factor Xa-digested proteins were then passed through the equilibrated amylose resin  column to collect Pro42-175 and Pro42-98, followed by the removal of factor Xa with factor Xa Cleaning Kit  according to the protocol described by the manufacturer. After factor Xa digestion of MBP-Pro42-98, size-exclusion gel chromatography  was used to purify Pro42-98 using a FPLC system.As described in detail previously , MBP-Pro°C, digestion by BMP-1 was examined by SDS-PAGE.Five μg of commercial MSTNpro (R&D Systems) proteins in 35 μL of 25 mM HEPES buffer (pH 7.5) containing 0.01% (v/v) Brij-35 were mixed with 25 ng of human BMP-1 (R&D Systems) in 5 μL of the same buffer. After incubation for 1 hr at 37E. coli (data not shown). The result of SDS-PAGE of amylose affinity-purified proteins in reduced and non-reduced conditions are shown in Four MBP-fused, truncated forms of pig MSTNpro proteins were initially expressed, including MBP-Pro42-218, MBP-Pro42-175, MBP-Pro42-115, and MBP-Pro42-98. As was demonstrated with the full sequence of MBP-fused pig MSTNpro (MBP-MSTNpro) in our previous study , all of 12-luciferase reporter gene assay  and MBP-Pro42-175 (2.7 nM) to suppress 1 nM MSTN activity were not significantly different from either that of commercial MSTNpro (2.2 nM) or full sequence MBP-MSTNpro (2.8 nM), indicating that residues 42–175 is sufficient to maintain full MSTN-inhibitory potency. The IC50 values of both MBP-Pro42-115 (17.7 nM) and MBP-Pro42-98 (24.1 nM) to suppress 1 nM MSTN activity were significantly higher than those of commercial MSTNpro or full sequence MBP-MSTNpro, indicating lower potencies of MBP-Pro42-115 and MBP-Pro42-98 than the MBP-MSTNpro in suppressing MSTN activity.All the MBP-MSTNpro proteins showed MSTN-inhibitory activity in the pGL3-(CAGA)ne assay . The IC550 values of full sequence MBP-MSTNpro to suppress 1 nM GDF11 (1.3 nM) was not different from that of commercial MSTNpro (1.2 nM). The IC50 values of MBP-Pro42-218 (6.6 nM) and MBP-Pro42-175 (9.7 nM) to suppress 1 nM GDF11 were not different from each other, but were significantly higher than that of full sequence MBP-MSTNpro (1.3 nM), indicating that unlike MSTN inhibition, MBP-Pro42-218 and MBP-Pro42-175 are not sufficient to maintain full GDF11-inhibitory potency. The IC50 value of MBP-Pro42-115 (19.0 nM) was significantly higher than those of MBP-Pro42-218 and MBP-Pro42-175, and the IC50 value of MBP-Pro42-98 (39.9 nM) was significantly higher than that of MBP-Pro42-115.Since GDF11 is highly related to MSTN with 90% amino acid sequence homology , 26, theTo examine whether the MSTN-inhibitory activities of MBP-MSTNpro proteins are associated with their bindings to MSTN, MSTN-binding of MBP-Pro42-175, MBP-Pro42-115, or MBP-Pro42-98 was examined in a pull-down assay. Since the MSTN-inhibitory capacity of MBP-Pro42-218 was not different from MBP-Pro42-175, the binding of MBP-Pro42-218 to MSTN was not examined. All three MBP-MSTNpro proteins showed their binding to MSTN . The intIn order to confirm the difference in Coomassie blue-stained band intensity of MSTN in the pull-down assay was due to differences in binding affinities of MBP-Pro42-175, MBP-Pro42-115 or MBP-Pro42-98 to MSTN, another pull-down experiment was performed. Five different concentrations  of MBP-Pro42-175 or MBP-Pro42-98 were mixed with 6 nM of MSTN in this pull-down experiment, and MSTN band intensity was examined by Western blotting with ECL method . As a neA previous study reported that a GST-fused human MSTNpro consisting of residues 42–98 showed no MSTN-inhibitory capacity , suggest50 value of Pro42-175 (44.9 nM) to suppress 1 nM MSTN was about 15 fold higher than that of MBP-Pro42-175 (2.7 nM), indicating that the presence of MBP as a fusion partner helped the binding of MBP-Pro42-175 to MSTN, leading to enhanced MSTN-inhibitory capacity of MBP-Pro42-175.The digestion of MBP-Pro42-175 led to appearance of two major bands of around 43 kDa and 15 kDa , indicatThe MBP-Pro42-98 was also incubated with factor X, and the digestion led to the appearance of two bands around 43 kDa and 7 kDa under reduced condition , indicatBMP-1 protease cleaves MSTNpro between Arg-98 and Asp-99 , 15, 20.The current study shows that a MBP-fused pig MSTNpro consisting of amino acid residues 42–175 is sufficient to maintain the full MSTN-inhibitory capacity. Our recent study also demonstrated that MBP-fused flatfish MSTN1pro domain consisting of residues 45–100 had a full MSTN-inhibitory capacity . Similarin vivo [The cleavage of MSTNpro between Arg-98 and Asp-99 by the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteinases and subsequent release of MSTN from the latent complex is known to be the mechanism for MSTN activation in vivo , 15, impin vivo  in whichin vivo –19, as win vivo , 18. Thein vivo , 30 indiin vivo –19.In summary, this study shows that MBP-fusion enhances the MSTN-inhibitory capacity of truncated forms of pig MSTNpro, and MBP-fused pig MSTNpro consisting of amino acid residues 42–175 is sufficient to maintain the full MSTN-inhibitory capacity.S1 Fig(A) After incubation of MBP-Pro24-98 with factor Xa, the total reaction mixture was centrifuged at 10,000 g for 3 min to soluble and in soluble fractions. Soluble fractions were subjected to 15% SDS-PAGE analysis under reduced (lane 1) and unreduced (lane 2) conditions, then visualized with Coomassie blue. *, Pro24-98. (B) The supernatant was subjected to gel filtration, and fractions 16, 19, and 22 were subjected to SDS-PAGE analysis under reduced condition. Arrow head indicate Pro24-98. (C) MSTN-inhibitory capacity of purified Pro24-98 (f16) was measured using the (CAGA) 12-luciferase reporter gene assay. The means of IC50 not sharing the same superscript are different at P<0.05.(JPG)Click here for additional data file.


We found that the oligopeptides 2 (Cys-Phe) and 5 (Cys-Asp) respectively presented IC50 values of 113 μM and 68 μM and also displayed favorable synergistic effects of the inhibitors in combination with ertapenem against genetic engineering-host E. coli BL21 (DE3)/pET30a-NDM-1 and a clinical isolate of P. aeruginosa with blaNDM-1. Flexible docking and partial charge study suggested the interaction between oligopeptide and NDM-1. Three types of action effects, hydrogen bond, electrostatic effect and π-π interaction, contributed to the inhibitory activities.‘Superbug’ bacteria producing NDM-1 enzyme causing wide public concern were first detected in a patient who visited India in 2008. It's an effective approach to combining β-lactam antibiotics with NDM-1 inhibitor for treating NDM-1 producing strain infection. In our research, we designed ten oligopeptides, tested IC Since penicillin was used in the treatment of bacterial infections, humans have always struggled against antimicrobial-resistant bacteria. It is the fact that many patients have died because of no effective anti-infection drugs –3. For tKlebsiella pneumoniae from India belonging to the subclass B1 MβLs superfamily [blaNDM-1 genes have appeared throughout the world [New Delhi metallo-β-lactmase-1 (NDM-1) was initially reported in erfamily . To datehe world . Recentlhe world –12, becahe world . NDM-1 ihe world . The cryhe world –17.In spite of the urgency for treatment of NDM-1 expressing bacteria, there has no much progress on exploring inhibitors for clinical use.50) was determined. Synergistic effect was also confirmed through combining these oligopeptides with ertapenem against genetically engineered bacterium producing NDM-1 and a clinical isolate of P. aeruginosa with blaNDM-1. The binding model suggested that hydrogen bond, electrostatic interaction between inhibitor and zinc ion, as well as π-π interaction in the active site of the enzyme played important roles in bioactivities.Based on the biochemical characterization of NDM-1 enzyme and molecular models of enzyme active center in the catalytic domain, oligopeptides were discovered to inhibit the hydrolytic activity of NDM-1, and the half maximal inhibitory concentration (ICE. coli BL21(DE3) cells were transformed with the pET30a plasmid encoding the NDM-1 gene (Gen Bank ID: HQ162469). E. coli BL21(DE3)/pET30a-NDM-1 was grown overnight at 37°C in liquid Luria-Bertani (LB) broth containing 50μg/ml kanamycin, 100μg/ml ampicillin and 50μM Zn(NO3)2 to select for E. coli clones containing the plasmid of pET30a-NDM-1. Two strains, E. coli BL21 (DE3)/pET30a-NDM-1 overexpressing NDM-1 and P. aeruginosa isolated from Renmin Hospital of Wuhan University with blaNDM-1 confirmed by PCR (data not shown), were cultured overnight, respectively. The cultures were diluted 1:100, grown to an optical density at 600 nm (OD600) of ~0.6, and spread onto LB agar. After naturally drying for five minutes, E-test strips  were applied to the ~150mm Mueller-Hinton (MH)  agar plates and grown for 24h at 35°C to 37°C without further intervention. The antibiotic strips respectively containing penicillin G, ampicillin, cephalothin, cefuroxime, ceftazidime, cefepime, meropenem, ertapenem, aztreonam were used for testing susceptibility. The results were obtained in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines [idelines .3)2 and 10mg/ml bovine serum albumin (BSA) preventing NDM-1 denaturation [max for ertapenem [50 values were determined more than three times by fitting the concentration dependence of residual enzyme activity to the nonlinear regression (curve fit) using GraphPad Prism .The full-length NDM-1 enzyme was expressed and purified by a method from a previously published procedure . All theturation . The ultrtapenem ,22. The E. coli BL21 (DE3)/pET30a-NDM-1 overexpressing NDM-1 and a clinical isolate of P. aeruginosa with blaNDM-1 were also utilized to evaluate the bioactivities of oligopeptides, which reversed NDM-1-mediated β-lactam antibiotics resistance. Determination of antibacterial synergy was performed using a serial twofold dilutions method recommended by CLSI guidelines [E. coli BL21 (DE3)/pET30a was used as a control in the experiment. Minimum inhibitory concentrations (MICs) of the agents were determined by the twofold serial dilution with Mueller-Hinton (MH) broth . For this assay, E. coli BL21 (DE3)/pET30a-NDM-1 was grown overnight in MH broth media containing 50μg/ml kanamycin at 37°C [E. coli BL21 (DE3)/pET30a-NDM-1. Every data of reading was repeated thrice, and each experiment mentioned above was performed at least two times.idelines . E. coli at 37°C . β-lacta at 37°C . The gro at 37°C . The lowMolecular docking is a commonly used and effective method in the research of binding model and structure-activity relationship to predict the interaction pattern between ligand and the target of known 3D-structure. Considering that the catalytic center was at the bottom of NDM-1 and many loops around it, flexible docking integrated in Discovery Studio version2.5 (DS v2.5)  was carried out to study binding model, which oligopeptides can be performed to force the target into alternative conformations . NDM-1 cE. coli BL21 (DE3)/pET30a-NDM-1, E. coli BL21 (DE3)/pET30a, E. coli BL21 and P. aeruginosa with blaNDM-1 are presented in E. coli BL21 (DE3)/pET30a and E. coli BL21(DE3) displayed the similar β-lactam antibiotic susceptibility, indicating that whether carrying an empty vector (pET30a) had no any effect on susceptibility of the host.The antimicrobial susceptibility patterns of E. coli BL21 (DE3)/pET30a-NDM-1 and P. aeruginosa with blaNDM-1 showed a decrease in susceptibility to penicillin G, ampicillin, cephalothin, cefuroxime, ceftazidime, cefepime, meropenem, ertapenem and aztreonam, demonstrating that the NDM-1 MβL can lead to β-lactam antibiotic resistance. The antimicrobial susceptibility pattern of the genetically engineered bacterium of E. coli BL21 (DE3)/pET30a-NDM-1 was also found to be similar to the clinical isolate of P. aeruginosa with blaNDM-1, with the MIC more than the maximum values of E-test antibiotic strips.50 values were 113 μM, 68 μM and 144 μM, respectively. Oligopeptides 1 (Cys-His), 4 , 6 (Cys-Gly-His) and 9  showed relatively weak NDM-1 inhibitory activities at a concentration range of 150–300 μM. Oligopeptides 3 (Cys-Tyr), 8 (Cys-Gly-Tyr) and 10 (Cys-Gly-Asp) displayed no obvious inhibitory effect below a concentration of 300 μM.The carbapenem antibiotic, ertapenem, was used as report substrate to determine the inhibitory activities of oligopeptides. According to the determination method mentioned above, all the tests repeated more than thrice. As shown in 50 = 68 μM). Comparing the structures of two oligopeptides 5 and 10, the difference is a glycine inserted in the middle. The increase of the glycine might lead to the loss of the inhibitory activity (IC50>300 μM). When other di-peptides 1 (IC50 = 182 μM), 2 (IC50 = 113 μM) and 4 (IC50 = 235 μM) were contrasted with the corresponding tri-peptides 6 (IC50 = 263 μM), 7 (IC50 = 144 μM) and 9 (IC50 = 249 μM), IC50 values indicated that the inhibitory activities appeared varying degrees of decline with one glycine inserted.Oligopeptide 5 containing the substructure of succinic acid and 3-mercaptopropionic acid had the best activity in all testing samples . The IC50 value of oligopeptide 4, which benzyl group of oligopeptide 2 was replaced by iso-propyl, displayed approximately a two-fold IC50 value of oligopeptide 2 . Analyzing the structure-activity relationship of oligopeptides 2–4, we found that the inhibitory effects against NDM-1 enzyme were positive correlation with the hydrophobic effect of side chain (hydrophobicity: benzyl group of oligopeptide 2 > iso-propyl group of oligopeptide 4 > p-hydroxybenzyl group of oligopeptide 3).The micro-structural differences between oligopeptides 2 and 3, the hydroxyl of benzyl group at the functional side chain, caused the huge difference of inhibitory effect (ICE. coli BL21 (DE3)/pET30a-NDM-1 overexpressing NDM-1 and P. aeruginosa with blaNDM-1 displayed high-level resistance to ertapenem. The strain was still able to grow in the broth media containing ertapenem at a concentration of 256 μg/ml. Oligopeptides 1, 2, 5 and 7, possessing IC50< 200μM, were chosen as representative inhibitors for determining synergistic antibacterial effects with ertapenem. None of the oligopeptides possessed any antibacterial activity within the concentration range of 25–200 μM. The MIC values of ertapenem in combination with inhibitory oligopeptides at various concentrations against E. coli BL21 (DE3)/pET30a-NDM-1 and the clinical isolate of P. aeruginosa were presented in 50 values. For the di-peptides 1, 2 and 5, the difference of oligopeptides sequence was the second amino acid residue, resulting in the diversity for synergistic effect. Oligopeptide in the presence of hydrophobic side chains had a better synergistic antibacterial performance, which was consistent with the IC50 values against NDM-1 enzyme. At the concentrations higher than 25 μM, oligopeptide 5 was capable to sensitize the NDM-1 producing strain to ertapenem at clinically achievable concentrations (MIC ≤ 128μg/ml) [E. coli BL21 (DE3)/pET30a-NDM-1were higher than the MIC values against the clinical isolate of P. aeruginosa. The reason might lie in the genetically engineered bacterium of E. coli BL21 (DE3)/pET30a-NDM-1 could over-express more NDM-1 enzyme than the clinical isolate of P. aeruginosa.It was obvious that all oligopeptides existed for synergies with ertapenem at a different level. Under the same concentration of the inhibitors, the MIC values of ertapenem exhibited uniform trend towards IC28μg/ml) . The triΔG) calculated by MM-PBSA along with the IC50 values of all oligopeptides were summarized in 50 values).To accurately simulate the induced-fit effect in the enzyme-inhibitor binding process, flexible docking was performed to explore the binding model. To characterize the binding affinities of docked complexes, binding free energies  was approximately one half of oligopeptide 2 (IC50 = 113μM).The binding model of oligopeptide 5 was shown in The results of inhibitory effect against NDM-1 enzyme proved that most of oligopeptides had inhibitory activities. Structure-activity relationship suggested that metal ion-binding group (such as sulfhydryl group and poly-carboxylic groups) and hydrophobic groups (such as benzyl group and iso-propyl group) were the pharmacophores significantly contributing to inhibitory activity.The synergistic antibacterial experiments disclosed that tri-peptides comparing to the di-peptides had no advantage of synergy. The insertion of glycine led to decreasing the binding strength between inhibitor and target.The established binding and interaction models demonstrated that hydrogen bond, electrostatic interaction and π-π interaction of parallel aromatic rings between the inhibitors and NDM-1 have played key roles in inhibiting the bioactivity of NDM-1.The two inhibitors, oligopeptides 2 and 5, possessed the best bioactivity in all testing inhibitors. For oligopeptide 2, there are three kinds of effects , while oligopeptide 5 only exists hydrogen bond and electrostatic interaction with target. We hold the opinion that if these three types of interactions achieve the optimal effectiveness, the chemical structure modification product of oligopeptides 2 and 5 may bring us an exciting bioactivity. Now, further chemical modifications of oligopeptide 2 are underway.


Aythya marila, Tufted Duck A. fuligula, Common Pochard A. ferina, Common Goldeneye Bucephala clangula and Eurasian Coot Fulica atra), and two are piscivores . Local changes at the level of particular species vary for different reasons. A local decline of the Common Pochard may simply be a consequence of its global decline. Climate change is responsible for some of the local changes in the study area, disproportionately favoring some duck species while being detrimental to others.Some species of birds react to climate change by reducing the distance they travel during migration. The Odra River Estuary in the Baltic Sea is important for wintering waterfowl and is where we investigated how waterbirds respond to freezing surface waters. The most abundant birds here comprise two ecological groups: bottom-feeders and piscivores. Numbers of all bottom-feeders, but not piscivores, were negatively correlated with the presence of ice. With ongoing global warming, this area is increasing in importance for bottom-feeders and decreasing for piscivores. The maximum range of ice cover in the Baltic Sea has a weak and negative effect on both groups of birds. Five of the seven target species are bottom-feeders (Greater Scaup  Mergellus albellus and Goosander Mergus merganser)  birds as a result of climate change . The disrganser) . This ha2, Dreissena polymorpha, which was introduced in the mid-19th century. By the 1960s, the biomass of zebra mussels in the Szczecin (Great) Lagoon was estimated at 110,000 metric tons , Świna Backward Delta, Kamień Lagoon, Dziwna Strait and Lake Dąbie (522.58 kmric tons . The disric tons . The ave05 kg/m2 . Fish arg others .Aythya marila—hereafter Scaup), Tufted Duck (A. fuligula), Common Pochard (A. ferina— hereafter Pochard), Common Goldeneye  and Eurasian Coot (Fulica atra—hereafter Coot); piscivores —Smew and Goosander —Greater Scaup  based on observations in other studies. Observations were always at a distance that does not disturb the birds in any way, and in Poland, this research required no ethical or scientific permits.Censuses were conducted using standard methods for non-breeding season waterbirds counts . Birds whttp://tutiempo.net). Another climate covariate was ice cover in the study area; data relating to this were published by the Polish Institute of Meteorology and Water Management. These data are from the observation point at Miroszewo on the shore of the Szczecin Lagoon . We compared the number of days with 100% ice cover in the period from 0 to 15 days prior to the bird counts. The ice cover of 100% refers specifically to the Miroszewo observation point. This estimate is a good approximation for the region. In practice, however, the ORE is never completely covered by ice  and after exclusion of minimal temperature VIF showed no multicollinearity issue between variables—ice cover (V IF = 1.04), max ice (V IF = 1.03), season (V IF = 1.03)—and these were used in the subsequent analyses. Frozen water by definition impedes bottom-feeding duck foraging, we are testing whether that pattern is changing in association with climate change. We used a general linear mixed model (GLMM) to test above-described relationship. The percentage of the flyway population present in the study area, estimated by species, was used as a target variable using the normal distribution response distribution and identity link function. Mixed models permitted repetition across survey months, methods  and species (random intercept). Thus, to test our assumptions we included the following interactions: feeding group*season, feeding group*ice cover and feeding group*max ice. Selection of the best model structure for the dependent variable was based on the Akaike information criterion (AIC)  in relation to flyway population for each species was used as our dependent variable . Thus, id by ice . Apart fon (AIC) . All poson (AIC)  and in oBelow, we present the results of our analyzes, the main one jointly examines two whole groups: piscivores and bottom-feeders and the second one where each species was analyzed. Bird numbers by feeding group were different in their relationships with ice cover; bottom-feeding species in the study area were more sensitive to lower temperatures and left sooner when colder weather increased ice cover, whereas a number of piscivorous species had a higher tolerance to the extent of ice cover . InteracScaup = 0.026 ± 0.010s.e., p = 0.011; however for Tufted Duck it was marginally insignificant β = 0.008 ± 0.004 s.e., p = 0.058), despite the general decline in their entire northern and western European populations; numbers of both species in the ORE were adversely affected by ice cover in that region  but not by ice cover in the whole Baltic . Relative numbers of Pochard in the ORE have declined , but so has the whole northern European population, abundance was negatively impacted by ice cover in both the study area ; and in the entire Baltic . For Goldeneye, the index for the ORE population was unchanged , despite the increase in the European population, abundance was negatively impacted by ice cover in the study area , but not by ice cover in the entire Baltic . Relative numbers of Coot in the ORE remained unchanged , despite the slight increase in the European population, abundance was negatively impacted by ice cover in both the study area , and in the entire Baltic . The ORE population index for Smew decreased , despite the increase in its flyway population, abundance in the ORE was unaffected by ice cover either in the study area , or in the Baltic as a whole . Finally, relative numbers of Goosander in the ORE remained unchanged , like those of the whole population wintering in north-western and central Europe; abundance in the ORE was unaffected by ice cover either in the study area  or in the Baltic as a whole . The details relating to all these species are listed in intercept = 82.011, s.e. = 28.49, βwinter =  − 00.40, s.e. = 0.0142, Chi-square = 8.001, df = 1, p = 0.005; Where particular species are concerned, the situation is more complex. The population indices of Scaup and Tufted Duck in the ORE exhibited an increasing trend , whereas Scaup remains almost exclusively in the ORE , it does feed on very small fish and on small invertebrates *100]; M, Count results; N, Size of population in a given season.A, date of count; B, season of count; C, species; D, month of count; E, number of days with 100% ice cover 15 days prior to count; F, mean daily temp. 15 days prior to count; G, mean max. temp. 15 days prior to count; H, mean min.temp.15 days prior to count; I, feeding group: B-bottom-feeders, P-piscivorous; J, count method: A - aerial, L - terrrestrial; K, maximum ice cover in the Baltic in thousand kmClick here for additional data file.10.7717/peerj.3604/supp-2Table S2The models were ranked using the Akaike information criterion (AIC). Δ AIC represents the difference between each model and the best-fit model. wi –Akaike weight (indicating model probabilities); df, –degrees of freedom. The terms in the models are represented by numbers: 1 –feed, 2 –ice cover, 3 –max ice, 4 –season, 5 –ice cover*feed, 6 –max ice*feed, 7 –season*feed.Click here for additional data file.10.7717/peerj.3604/supp-3Table S3The parameters show the interaction between season and species, ice cover and species, max ice and species. The interaction parameters species*season, species*ice cover, species*max ice were used to predict the values presented in Click here for additional data file.10.7717/peerj.3604/supp-4Table S4(1) Target species, (2) trend of flyway population after Click here for additional data file.


Mycoplasma pneumoniae is strongly associated with new onset asthma and asthma exacerbations. Until recently, the molecular mechanisms utilized by M. pneumoniae to influence asthma symptoms were unknown. However, we recently reported that an ADP-ribosylating and vacuolating toxin called the Community Acquired Respiratory Distress Syndrome toxin, CARDS toxin, produced by M. pneumoniae was sufficient to promote allergic inflammation and asthma-like disease in mice. A mouse model of CARDS toxin exposure was used to evaluate total and CARDS-toxin specific serum IgE responses. Mast cell sensitization, challenge, and degranulation studies determined functionality of the CARDS toxin-specific IgE. In the current study, we report that a single mucosal exposure to CARDS toxin was sufficient to increase total serum IgE and CARDS toxin-specific IgE in mice. Mice given a second mucosal challenge of CARDS toxin responded with significant increases in total and CARDS toxin-specific IgE. CARDS toxin-specific IgE bound to an N-terminal peptide of CARDS toxin but not the C-terminal peptide. Likewise, full-length CARDS toxin and the N-terminal peptide induced mast cell degranulation. Altogether, these data demonstrate that exposure to CARDS toxin is sufficient to generate functional IgE in mice. M. pneumoniae and CARDS toxin are strongly associated with asthma exacerbations raising the possibility that the CARDS toxin-specific IgE-mast cell axis contributes to disease pathogenesis. Asthma and allergic diseases remain a significant source of morbidity and mortality in the developed world . This isMycoplasma pneumoniae is of particular interest due to prevalence in the community, the seasonal nature of infections, and the rapidly increasing rates of macrolide resistance in M. pneumonia [M. pneumoniae is the leading cause of community acquired pneumonia amongst children in the US [M. pneumoniae colonizes tracheal and bronchial epithelium causing cytotoxicity characterized by loss of ciliary function and epithelial vacuolation [Of the atypical pathogens, neumonia –13 Curren the US . Dependin the US , 14. M. uolation , 16.Mycoplasma pneumoniae has strong clinical associations with asthma exacerbations and morbidity in both children and adults [M. pneumoniae, the Community Acquired Respiratory Distress Syndrome toxin (CARDS toxin), was identified [M. pneumoniae infection [d adults , 13, 17.entified , 19. CARnfection , 20–24. nfection , 20, chanfection , 20. AddMycoplasma pneumoniae infection is strongly linked to exacerbations of asthma in children and adults[nd adults, 25, 26.nd adults, 27, sugAlthough many of the mechanisms leading to allergic inflammation remain poorly defined, the immunoglobulin-E (IgE) and mast cell axis are key mediators of the allergic reaction . In animMycoplasmas, and viruses [Staphylococci and Streptococci [Classically, mast cells and IgE are considered protective against parasitic infections. We now appreciate that mast cells have a broader role in immunity providing protection against Gram-negative bacteria,  viruses –39. Likeptococci –42. Inteptococci –45. Whilptococci , there iIn this study we utilized our mouse model of CARDS toxin-associated asthma to test the hypothesis that exposure to CARDS toxin leads to the generation of a functional CARDS toxin-specific IgE.5-week-old BALB/cJ mice were purchased from Jackson Laboratory  and STAT6-/- mice on the BALB/c background were provided by Dr. Michael Berton and bred in house. All mice were maintained in an AAALAC-approved facility with all animal work conducted according to relevant national and international guidelines. Work was conducted under a protocol approved by the University of Texas Health Science Center at San Antonio Institutional Animal Care and Use Committee (protocol #12070X) established. During experimental procedures, animals were monitored by laboratory staff twice a day and there were no apparent animal illnesses nor unexpected deaths during this study. Animal suffering was minimized by providing free access to food and water, animals had soft bedding for the duration of the experiments and all procedures were done under general anesthesia, 3% isoflurane and oxygen.Bone marrow was collected from BALB/c mice and incubated in complete bone marrow mast cell (BMMC) media and incubated four weeks using a modified protocol previously described . BMMC meRecombinant CARDS toxin was kindly provided by Drs. Joel Baseman and TR Kannan. Briefly, rCARDS toxin was expressed and purified as previously described in detail , 20 and M. pneumoniae.Mice were sedated with 3% isoflurane in air supplemented with oxygen. Seven hundred pmol of rCARDS toxin was administered IT via forced oropharyngeal aspiration as we have described . The dosSerum was allowed to clot at room temperature and stored overnight at 4°C. Samples were then centrifuged at 14000 rpm for 15 minutes at 4°C. Serum was collected and stored at -80°C.3PO4 stop solution. Data represents two independent assays measured in triplicate.To measure total IgE, serum samples were diluted at 1:50 and assayed using a commercially available kit  according to the manufacturer’s instructions. rCARDS toxin-specific IgE was determined with a modified ELISA protocol previously described . Briefly2 μg of full-length rCARDS toxin, an amino terminus fragment (amino acids 1–249), a carboxy terminus fragment (amino acids 261–591), and ovalbumin were run on a 10% SDS PAGE gel. Gels were transferred to PVDF membranes  using a semi-dry transfer Owl apparatus . Membranes were blocked with 5% milk in TBS with 0.1% tween-20. Membranes were probed with serum from rCARDS toxin treated mice overnight at 4°C. Membranes were washed with TBS + 0.1% tween-20 and incubated with anti-mouse IgE cross-adsorbed to IgM and IgG in 5% milk in TBS with 0.1% tween 20 at room temperature for 2 hours. Membranes were washed and probed with a chicken anti-goat-HRP for 1 hour at room temperature. Membranes were washed and developed with enhanced luminol reagent  using autoradiography.6. Mouse serum was not depleted of non-IgE immunoglobulins prior to sensitization. Cells were washed in Tyrode’s buffer  and resuspended in Tyrode’s buffer containing 10% deuterium to a density of 50,000 cell/well in a 96 well plate. Cells were challenged with full-length rCARDS toxin, rCARDS toxin fragments or controls and incubated 45 minutes. Cells were centrifuged and supernatant added to p-nitrophenyl N-acetyl-β-D-glucosamide (PNAG)  (3.5 mg/ml) dissolved in citrated buffer  to measure β-hexosaminidase activity. The cell pellet was lysed in Tyrode’s buffer with 0.1% Triton-X to measure total degranulation and incubated with PNAG. Supernatant and cell lysates were incubated at 37°C for 90 minutes. Reactions were stopped with 400 mM glycine pH 10.7 and plates read at 405 nm with a 620 nm reference filter. Percent hexosaminidase release was calculated as OD of the supernatant/ (OD of the supernatant + OD of the lysed cellular pellet) X100.A modified version of a previously described protocol was used . Brieflyt-test using GraphPad Prism version 5.04 .Experiments were repeated a minimum of 2 times with 6–10 animals, depending on the specific protocol (except where noted). All results were expressed as the mean ± S.D. Statistical differences were determined using a two-way ANOVA with Bonferroni post-hoc test or two-tailed Student We previously reported that a single exposure to CARDS toxin results in prolonged changes in lung histopathology that lasted up to 56 days post-exposure . FurtherPreviously, we reported that heat-inactivated (HI) CARDS toxin does not induce the cytokine responses nor the characteristic changes in pulmonary histopathology observed with exposures to active toxin . To testTo test the ability of CARDS toxin to boost total serum IgE responses, mice were treated IT with CARDS toxin and then 21 days later mice received a second IT dose of CARDS TX. Total serum IgE concentrations were determined by ELISA on days 2, 4, 7, and 10 after treatment with the second dose of CARDS toxin. Following the second exposure to CARDS toxin, there was a rapid rise in total serum IgE evident by day 2 after-exposure that peaks at 10-fold over vehicle controls on days 7 and 10 after- exposure (p<0.005) .A single mucosal exposure of mice to CARDS toxin resulted in increased total serum IgE suggesting the production of CARDS toxin-specific IgE. As shown in To partially define the region of CARDS toxin recognized by CARDS toxin-specific IgE, we tested immuno-reactivity in Western blots. Briefly, equimolar amounts of full length CARDS toxin, a 1–249 amino acid N-terminal fragment, a 261–591 amino acid C-terminal fragment, and chicken ovalbumin (negative control) were fractionated by gel electrophoresis and transferred to a PVDF membrane. The membrane was probed with pooled sera containing high titer anti-CARDS toxin-specific IgE. Immunoreactive bands were detected with an anti-mouse IgE secondary antibody and visualized with chemiluminescence. As shown in To test if CARDS toxin-specific IgE was functional, mast cells were differentiated from mouse bone marrow (BMMC). BMMCs were then sensitized with pooled sera containing high titer anti-CARDS toxin IgE. As controls, BMMCs were sensitized with sera from vehicle control mice or untreated mice. Sensitized and non-sensitized mast cells were then challenged with different versions of CARDS toxin or nonspecific activators and hexoaminodase activity in the cell culture supernatants measured as an indicator of degranulation. Mast cells sensitized with serum containing anti-CARDS toxin-specific IgE and challenged with full length or the N-terminus of CARDS toxin resulted in 17–19% hexosaminidase release . ImportaMycoplasma pneumoniae, respiratory syncytial virus, rhinovirus, and exposure to molds are known to enhance cytokine responses that promote allergic inflammation [Allergic diseases remain a significant source of morbidity and in the case of anaphylaxis, risk of mortality. Infection is a significant source of exacerbation in allergic asthma but the mechanisms underlying pathogen-associated disease worsening are poorly understood. Infections with ammation , 17, 52.We previously showed that CARDS toxin could induce allergic-like inflammation in BALB/c mice after a single challenge. This was defined by mucus hypersecretion, increased Th2 responses, eosinophilia, and airway hyperreactivity . AdditioThe C-terminal peptide of CARDS toxin did not react in immunoblots to sera containing CARDS toxin-specific IgE. Likewise, the C-terminal peptide of CARDS toxin did not result in mast cell degranulation when used to challenge sensitized mast cells. These results could imply that the majority of IgE epitopes are exclusively in the N-terminus of CARDS toxin. However, we cannot rule out a contribution of conformational or linear epitopes in the C-terminus that do not respond in our assays due to inappropriate protein folding. Further, we only used full length CARDS toxin to immunize animals and thus, did not test the ability of N- or C- terminal fragments to induce toxin-specific IgE directly. We did demonstrate that both heat inactivated toxin and the CARDS toxin E132A mutant do not induce a strong IgE response suggesting that the enzymatic activities of the toxin are required strengthening the suggestion that full-length toxin is required for optimal IgE responses.M. pneumoniae infections are associated with increased total IgE, however it was unclear if CARDS toxin could replicate this aspect of M. pneumoniae infections [M. pneumoniae or RSV infections [fections . IgE is fections . In atopfections –56. In tfections . Likewisfections –59. CurrM. pneumoniae CARDS toxin to induce an IgE response and its potential to function as a classical allergen.Further, many environmental allergens associated with asthma exacerbations including house dust mites, cockroaches, fungi and many pollens  are knowM. pneumoniae infection [Mast cells are important to protect mice from nfection . Howevernfection –37, 39. nfection , 32. At Staphylococcal and streptococcal enterotoxins are potent inducers of IgE and toxin-specific IgE and represent the best-studied group of IgE-associated bacterial toxins [Although many proteins have been identified as allergenic, bacterial toxins are not common allergens. l toxins –42. Unlil toxins , 51. TheThe mast cell-IgE axis is central to allergic inflammation. A series of signaling events occurs when mast cell surface bound IgE is cross-linked in the presence of allergen, leading to cytokine secretion, de novo synthesis of leukotrienes and mediator release/degranulation by mast cells . These gThe presence of CARDS toxin-specific IgE in mice that can lead to IgE-dependent mediator release, suggests the possibility that CARDS toxin could be acting as an allergen in human asthma. While this remains to be determined, these findings add to rapidly accumulating evidence that CARDS toxin is a significant virulence factor with the potential to contribute to significant pathology in experimental and human asthma.


Aplysia californica participates in several forms of presynaptic plasticity including homosynaptic depression, heterosynaptic depression, facilitation and the reversal of depression. The calcium channel triggering neurotransmitter release at most synapses is CaV2, consisting of the pore forming α1 subunit (CaV2α1), and auxiliary CaVβ, and CaVα2δ subunits. To determine the role of the CaV2 channel in presynaptic plasticity in Aplysia, we cloned Aplysia CaV2α1, CaVβ, and CaVα2δ and over-expressed the proteins in Aplysia sensory neurons (SN). We show expression of exogenous CaV2α1 in the neurites of cultured Aplysia SN. One proposed mechanism for heterosynaptic depression in Aplysia is through inhibition of CaV2. Here, we demonstrate that heterosynaptic depression of the CaV2 calcium current is inhibited when a channel with a Y-F mutation at the conserved Src phosphorylation site is expressed, showing the strong conservation of this mechanism over evolution. We also show that the Y-F mutation reduces heterosynaptic inhibition of neurotransmitter release, highlighting the physiological importance of this mechanism for the regulation of synaptic efficacy. These results also demonstrate our ability to replace endogenous CaV2 channels with recombinant channels allowing future examination of the structure function relationship of CaV2 in the regulation of transmitter release in this system.The sensory neuron of  Aplysia pleural sensory neurons are body wall sensory neurons involved in the defensive withdrawal reflexes of the animal and the locus for a variety of forms of synaptic plasticity2. Serotonin released with a noxious stimulus increases sensory neuron excitability and transmitter release5. Conversely, dopamine, FMRFamide, and activation of 5HT1 receptors reduce sensory neuron excitability and transmitter release leading to heterosynaptic depression11. As is the case at most synapses, transmitter release at Aplysia sensory neurons is triggered by voltage-gated calcium entry through CaV2 calcium channels6. Aplysia pleural sensory neurons in culture have both CaV1 and CaV2 high-voltage-activated (HVA) currents and do not appear to express a low-voltage-activated calcium current (CaV3 or NaV2), allowing isolation of the CaV2 current with the block of the CaV1 current with nifedipine11. With the exception of the vertebrates, bilaterians have a single gene coding the pore forming CaV2 alpha 1 subunit (CaV2α1). As there is evidence that the Aplysia CaV2 current is regulated by dopamine11, FMRFamide6, and bidirectionally with serotonin (depending on the identity of the 5HT receptors activated12) we cloned the Aplysia CaV2α1 along with the accessory subunits CaVβ and CaVα2δ to further investigate this modulation.V2 calcium current with G-protein coupled receptor (GPCR) activation is well documented. The G-protein Gβγ subunit can inhibit the channel directly through a thoroughly studied mechanism termed voltage-dependent (VD)-inhibition as the inhibition can be relieved with strong membrane depolarization13. However, VD-inhibition does not appear to occur with invertebrate CaV2 channels14. Rapid CaV2 inhibition can also occur with GPCR activation through downstream Src kinase activity16. Src kinase phosphorylation of the CaV2α1 C-terminal EF-hand Y1747 is involved in the voltage-independent (VI)-inhibition with μ-opioid receptor activation18 and with GABAB receptor activation19. Here we show that the Y1747 residue is highly conserved, found in most CaV2 sequences, and point mutation of the Y1747 ortholog to phenylalanine in the Aplysia CaV2α1 (Y1501F) reduces the inhibition of the calcium transient with dopamine and 5HT1A activation measured with fluorescence imaging. Furthermore, CaV2α1 Y1501F expression in presynaptic sensory neurons reduced the heterosynaptic depression at sensory to motor neuron synapses with 5HT1A activation. This indicates that the VI-inhibition of the CaV2 current through Src kinase phosphorylation of the F-helix EF-hand of CaV2α1 is a physiologically important and highly conserved mechanism of CaV2 regulation.The inhibition of the CaV2α1 subunits in cultured sensory neurons required at least 48 h of expression, evidenced by the inability of the Y1501F mutant to affect the inhibition of 5HT1A activation with only 24 h expression. The block of the inhibition with Y1501F expression appears to be complete at 48 h, indicating near complete substitution of the endogenous alpha 1 subunits with recombinant subunits at this time point.Functional expression of the exogenous, RFP-tagged CaV2 calcium channel from Aplysia californica  using primers designed from searching the Aplysia transcriptosome (www.aplysiagenetools.org) for sequences with homology to the cloned Lymnaea CaV2 channel. Partial sequences were identified on distinct transcripts, however, several highly repetitive and CG rich regions probably prevented assembly of the entire message. Despite this, we were able to assemble a complete CaV2α1 using PCR  and CaVα2δ subunits (KY705238) sequences. Comparing the Aplysia CaV2α1 sequence to the available genomic and transcriptome data for Aplysia, reveals at least five sites with alternate sequences , in other Molluscs , and in some cases in Chordates as well based on transcriptome data and previous publications .Electrophysiological examination of sensory neurons expressing RFP-Caity Fig. . SNs expold Fig. . This chity Fig. . This coV2 calcium current in Aplysia sensory neurons11. The inhibition through 5HT1A activation was blocked with the Src kinase inhibitor PP2, consistent with observations of voltage-independent inhibition of vertebrate channels occurring through Src kinase modulation11. This voltage-independent inhibition occurs in mammalian CaV2.2 channels expressing exon e37a through tyrosine kinase phosphorylation of the EF-hand Y174715. The tyrosine residue in the F-helix of the C-terminal EF-hand of CaV2 is conserved in the Aplysia CaV2α1 sequence, and comparison to other metazoan high-voltage activated calcium channels reveals that this residue is highly conserved throughout the evolution of the CaV2α1 subunit . The first evolutionary branch to have a recognizable distinction between CaV1 and CaV2 are Cnidarians, and Cnidarians have multiple CaV2 channels, some of which contain the tyrosine  and mammalian CaV2.2 channels expressing e37b. This data suggests that the Y of the F-helix of the CaV2α1 C-terminal EF-hand is highly conserved throughout evolution and may underlie a conserved mechanism for modulation of CaV2 currents and thus downstream evoked transmitter release. To test the hypothesis that the Src kinase regulation of Aplysia CaV2 occurs through the Aplysia ortholog of the F-helix EF hand tyrosine, we generated a non-phosphorylatable mutant Aplysia RFP-CaV2α1 Y1501F.We have previously shown that dopamine acting through an endogenous receptor and 5HT acting through expressed 5HT1A receptors inhibits the endogenous CaV2α1 subunit and the Y/F mutant both express on the cell surface and conduct a current consistent with a CaV2 current we subcloned our channels subunits into vectors suitable for expression in HEK293t cells, a cell line derived from human embryonic kidney cells. Either Aplysia RFP-CaV2α1 wt or Aplysia RFP-CaV2α1 Y1501F expression along with the Aplysia accessory subunits Aplysia CaVβ and Aplysia CaVα2δ generated inward barium currents consistent with other CaV2 currents, including isolated endogenous Aplysia CaV2 currents . The activation, inactivation and steady-state inactivation curves are similar for both wildtype and Y1501F mutant, thus both constructs code functional channels with similar kinetics that can express on the cell surface . While incubation at 28 °C doubled whole-cell current density, the measured currents were still very weak. This may relate to the natural operating temperature of the channels in the animal (~14 °C), which is much lower than that of other CaV2 channels expressed in HEK cells. The activation, inactivation, and steady-state inactivation parameters of RFP-tagged wildtype and RFP-tagged Y1501F mutant channels expressed in HEK cells demonstrates that both of our constructs can produce functional CaV2 channels and that the Y1501F mutation does not by itself cause a significant change in the properties of the CaV2 channel.To ensure that the RFP-tagged wildtype Ca11. Thus, we used fluorescent calcium imaging to measure the calcium current. Despite the increased RFP expression seen at 48 h, expression of RFP-CaV2α1 did not affect the peak amplitude of the Fluo-4 transients in the neurites , despite similar RFP expression with the two constructs . The absence of a change in the Fluo-4 transient after expression of the channel could be due to the lack of surface expression of the exogenous calcium channel or constraints on calcium channel surface expression in this system. If there are constraints on calcium channel expression, then it is conceivable that the exogenous CaV2α1 subunits might replace the endogenous CaV2α1 subunits in the surface expressed CaV2 channel. To explore this possibility, we compared the amount of heterosynaptic inhibition of the Fluo-4 transients without expressing a recombinant CaV2α1, expressing RFP-CaV2α1, and expressing RFP-CaV2α1 Y1501F. Since in our previous study, only about 50% of pleural sensory neurons in culture respond to endogenous modulators11, we co-expressed the Aplysia 5HT1A receptor9. While, the Aplysia 5HT1A receptor is endogenously expressed in a very small percentage of pleural sensory neurons (~5–10%22), it can be exogenously expressed and subsequently activated with the agonist 8-OH-DPAT which strongly and consistently reduces both membrane excitability and the endogenous CaV2 current, reducing variability in this experiment11. Inhibition of the Aplysia CaV2 current with 5HT1A expression and activation was also found to be sensitive to the Src kinase inhibitor, PP211. Thus, we compared the inhibition of the Fluo-4 transient with 5HT1A expression/activation in sensory neurons either with RFP-CaV2α1 or the point mutant RFP-CaV2α1 Y1501F. Application of the 5HT1A agonist 8-OH-DPAT without expression of 5HT1A (No Inj) show only a slight reduction in current. In contrast, when 5HT1A was expressed alone (1A) or with RFP-CaV2α1 WT (wt + 1A), 8-OH-DPAT caused a statistically significant ~50% reduction in the peak single action potential Fluo-4 transient  or the Src inhibitor PP2 was present (wt + 1A + PP2), no significant inhibition was seen. This suggests that at least a portion of the 5HT1A inhibition of the CaV2 current is through Src kinase, and requires a tyrosine in the F-helix of the EF-hand of CaV2α1.The time required for cell adhesion to the dish, plasmid injection, and subsequent expression of the CaV2 constructs results in neurite outgrowth that is problematic for achieving an effective space clamp required for voltage-clamp analysistes Fig. . Althougent Fig. . In contV2α1 subunits reduces initial membrane excitability as measured with the amount of current required to reach action potential threshold. The three groups expressing exogenous CaV2α1 subunits for 48 h  required more depolarizing current to reach action potential threshold before addition of agonist  .As described in Fig. ps) Fig. . InteresV2α1 Y1501F + 5HT1A groups is a negative effect of the mutation on 5HT1A surface expression or signalling downstream of the receptor. An additional effect of signalling through 5HT1A is a decrease in membrane excitability. Indeed, the amount of current to reach action potential threshold was increased with application of the 5HT1A agonist 8-OH-DPAT, reducing membrane excitability in all groups injected with 5HT1A, confirming activation and signal transduction of the exogenously expressed 5HT1A receptors, and no effect of the Y1501F mutation on this expression  allowing visualization of the 5HT1A expression  amplitude, a feature of the sensory to motor neuron synapse that is independent of heterosynaptic signalling23. The greatest amount of homosynaptic depression happens between PSP1 and PSP2, and this was not significantly affected with expression of either eGFP-5HT1A or the CaV2α1 subunits   Fig. . This suAmphimedon and the comb jellyfishes Mnemiopsis and Pleurobrachia have a single gene coding the alpha 1 subunit of a high voltage-activated calcium channel as does the sister group to the Metazoans, the Choanoflagellates. In well sequenced Cnidarian genomes there is a clear CaV1 gene and many CaV2 genes, thus CaV1 and CaV2 diverged before the Cnidarian branch and early in Cnidarian evolution the CaV2 gene was then duplicated multiple times24. All well sequenced Bilaterians have a single CaV1 gene and a single CaV2, with the exception of the vertebrates where genome duplication has led to four CaV1 and three CaV2 genes26. The tyrosine at 1747 first appears in Cnidarians, where many of the Cnidarian CaV2α1 sequences have the conserved tyrosine. Y1747 (Y1501 in Aplysia) is conserved in most Bilaterian CaV2α1 subunits with very few exceptions. Thus, the F-helix EF-hand tyrosine is highly conserved, a feature of most CaV2α1 subunits.Early Metazoans such as the sponge 19. The Src kinase-mediated inhibition of the N-type calcium current can be regulated in mammalian neurons through the mutually exclusive alternate exon pair e37a and e37b, where the F1747 containing e37b channels are insensitive to Src kinase inhibition17. The first indication that this may represent a conserved mechanism was our demonstration that the strong and consistent VI-inhibition of the Aplysia CaV2 current with activation of the 5HT1A receptor is blocked with a Src kinase inhibitor11. Here we show that this inhibition requires the equivalent tyrosine as Src-kinase dependent inhibition of the mammalian channel. The similarity in the extent of the block of 5HT1A inhibition of the single action potential Fluo-4 transient with PP2 and Y1501F suggests a few points of note. Firstly, it suggests that our over-expressed point mutated alpha 1 subunits are getting out to the plasma membrane participating in voltage-mediated calcium entry and largely replacing endogenous CaV2α1 at 48 h, a quality that is not measurable with the N-terminal RFP tag. Secondly, the inhibition of the Aplysia CaV2 current in sensory neurons mediated by Src kinase activity requires CaV2α1 Y1501. This is most likely through phosphorylation of this residue by Src, but at present we lack the reagents to directly measure phosphorylation of this residue and its dependence on Src. Expression of CaV2α1 Y1501F not only prevented inhibition of calcium currents with dopamine and 5HT1A activation in isolated sensory neurons, but also reduced the decrease in transmitter release mediated by 5HT1A receptor activation at sensory-motor neuron synapses. The partial reversal of heterosynaptic depression with expression of CaV2α1 Y1501 indicates that the heterosynaptic inhibition of transmitter release, at least partly, occurs through the inhibition of the CaV2 current. Our experiments cannot rule out additional mechanisms independent of CaV2α1 Y1501 that also contribute to heterosynaptic depression. Previous examination of heterosynaptic depression with FMRFamide implicated p38 MAP kinase and arachidonic acid in heterosynaptic depression28, while the mechanisms of this inhibition are not understood, similar pathways may combine with CaV2 inhibition to mediate heterosynaptic depression with 5HT1A activation or alternatively, be involved in the activation of Src. The strong conservation of the Y1501 residue in most CaV2 alpha 1 subunits with very few exceptions implies that this mechanism is ancient and we would predict that even Cnidarian channels could be regulated through this mechanism.The phosphorylation of Y1747 of vertebrate calcium channels with Src kinase leads to rapid VI-inhibition of the current22), but RFP-CaV2α1 Y1501F is not effective at preventing the inhibition of the calcium current at this same time point. The change in efficacy of RFP-CaV2α1 Y1501F on Fluo-4 inhibition with the increase in the duration of expression from 24 h to 48 h also coincides with an RFP signal in the distal neurites and a change in membrane excitability. This indicates that the functional expression of CaV2α1 requires additional time, potentially a result of the highly regulated trafficking and surface expression previously noted for these channels30. Consistent with the somatic exclusion of CaV2 channels in Aplysia sensory neurons11, the action potential Fluo-4 transients of expressed recombinant RFP-CaV2α1 subunits with the CaV1 blocker nifedipine are only measured in the neurites despite the strong RFP signal in the soma. Whatever the mechanism that allows somatic expression of CaV1α1 but excludes CaV2α1 is not overwhelmed with CaV2α1 over-expression. The coincident change in membrane excitability observed at all SN expressing an exogenous CaV2α1 protein expressed for at least 48 h appears to relate to CaV2α1 surface expression, however the mechanism for this is uncertain. CaV2 channels should not open near SN resting potentials (and SNs are not spontaneously active neurons). Nor does it appear to be related to increased overall surface expression of CaV2 channels with expression of recombinant channels since the initial Fluo-4 transients were not increased with CaV2α1 expression  as suggested in other systems35, endogenous accessory subunits  appear sufficient for the substitution of the endogenous/exogenous alpha 1 subunits as the efficacy of Y1501F mutant was similar with both 5HT1A inhibition  and dopamine inhibition . This is further supported by the change in membrane excitability and distal neurite RFP expression with 48 h expression of CaV2α1 isoforms that was also unaffected by coexpression of the CaVβ and CaVα2δ subunits. Thus, the factors that tightly regulate and limit CaV2 surface expression involve more than just subunit availability in these neurons.While we attempted to enhance RFP-CaV2α1 Y1501F expression consistently resulted in weaker initial single action potential Fluo-4 transients even though the Fluo-4 loads were similar between groups and experiments  also has differences from the e37a isoform that reduces current amplitudes through changes in channel gating kinetics36. Perhaps the change in CaV2.2e37b channel kinetics are mimicked with phosphorylation of Y1501, or the inhibition through phosphoregulation could occur through production of a new binding domain such as an SH2 or PTB, that leads to the loss or gain in a regulatory binding partner. The proximal CaV2α1 C-terminal is known to be a site of channel kinetic regulation as the Drosophila CaV2 paralytic mutation (cac ts2) occurs through a single amino acid point mutation 9 amino acids downstream of Y1501 by changing channel kinetics44. Furthermore, the CaV1 calcium-dependent inhibition involves the EF-hand, as does the voltage-dependent inhibition through intracellular Mg2+, further highlighting the potential of this proximal C-terminal domains involvement in altering channel kinetics47. Further experiments will be needed to understand the mechanisms underlying the inhibition of the CaV2 current with Src kinase. Given the many potential roles of Y1501, in surface expression, channel gating kinetics, and phosphoregulation, it remains unclear which of these features has led to the strong evolutionary conservation of this residue.Cants Fig. . While tAplysia CaV2 calcium channel demonstrates that we can functionally replace endogenous CaV2α1 subunits with recombinant proteins and confirms that the CaV2α1 subunit EF-hand tyrosine Y1501 (orthologous to rat N-type Y1747) is a target for regulation by GPCRs through Src. These experiments also demonstrate that the inhibition of the CaV2 calcium current is at least partially responsible for the inhibition of neurotransmitter release with heterosynaptic depression.Overall, this initial work using the cloned V2α1 was assembled from transcriptome data (www.aplysiagenetools.org) based on finding transcripts with homology to the Lymnaea CaV2α1 sequence48. No transcript in this database crossed two large GC rich regions . We first cloned the 5′ end of CaV2.2 from the start codon until the I-II llinker (1.2 kB) into pNEXmRFP to generate a fusion protein with monomeric RFP attached to the cytoplasmic N-terminal of CaV2α1. The 5′ end was cloned with primers containing a KpN I site at the 5′ end and a BlpI site at the 3′ end to allow cloning into pNEX3mRFP (Table A tentative sequence of CaV2.2 and with a 3′ BlpI site to insert them into the growing CaV2.2 sequence (PCR primers are listed in table I). For the GC region in the large C-terminal splice (poly glycine stretch) we used nested PCR and then assembled the sequence in pJET2.1 before inserting back into the growing CaV2.2 sequence. For the region containing the Src phosphorylation site, we first cloned the PCR fragment into pJET 2.1 (Thermo Fisher Scientific) and then used a primer containing the adjoining BamHI site to generate the tyrosine (Y) to phenylalanine (F) mutation , RFP-CaV2α1-ΔeCT, RFP-CaV2α1-ΔeCT(Y1501F). For all the results shown, results with splice and no splice were combined as no significant differences were seen in expression, effects on excitability, or inhibition by modulatory inputs.We then sequentially used PCR to clone fragments overlapping at their 5′ end with a unique restriction site in CaV beta subunit (CaVβ) and alpha2-delta subunits (CaVα2δ) were identified from transcriptome data (www.aplysiagenetools.org) using homology to the Crassostrea and Lottia sequences. For the CaVβ subunit, two PCR pieces . The joining site is identical since pNEX3-mRFP was generated from pCDNA3-mRFP. CaVβ and Cavα2δ were excised from pNEX3 with HindIII and KpnI and inserted into pCDNA3 at the same sites. Joinings were confirmed by sequencing.To express the channel in HEK293t cells, the channel was excised from pNEX RFP- CaV1.2 XP_008761438.1, Lymnaea s.CaV1 AAO83839.1, Nematostella v. CaV1 XP_001639054.1, Salpingoeca r. XP_004989719.1, Amphimedon q. XP_011406227.1, Mnemiopsis l. AEF59084.1, Trichoplax a. s1 XP_002108930.1, Trichoplax a. s2 XP_002109775.1, Nematostella v. 2a ABAV01003013.1, Nematostella v. 2b ABAV01007705.1, Nematostella v. 2c ABAV01002357.1, Schistosoma m. AAK84311.1, Lymnaea s. AAO83841.1, Capitella t. 51958, Drosophila m. NP_001245638.1, Caenorhabditis e. NP_741734.1, Strongylocentrotus p. XP_011662956.1, Branchiostoma f. AAM18875.1, Ciona i. XP_018670105.1, Petromyzon m. ABQ96268.1, Danio r. 2.1 XP_017210285.1, Danio r. 2.2 XP_009299779.1, Danio r. 2.3 XP_017212913.1, Gallus g. 2.2e37a XP_015134748.1, Gallus g. 2.2e37b XP_015134761.1, Rattus n. 2.2e37a XP_008759800.1, Rattus n. 2.2e37b XP_006233631.1.Accession numbers for the sequences displayed in Fig. 49. Cells were transiently transfected with RFP-CaV2α1 or RFP-CaV2α1 (Y1501F), along with Aplysia CaVβ and CaVα2δ at a plasmid ratio of 1:2:2 to increase the chances that RFP fluorescent cells also express the accessory subunits. Transfections were carried out using Polyfect (Qiagen) according to the manufacturer’s instructions. 24 h following transfection, cells were trypsinized and re-plated at a confluence of 30,000 to 60,000 cells per recording chamber dish and transferred to a 5% CO2 incubator at 28 °C for 48–96 h before recording. For electrophysiological recordings, the extracellular solution was composed of in mM: . Whole-cell pipettes were fabricated to have resistances of 3–10 MΩ when filled with the internal solution composed of in mM: . Cells with RFP fluorescence were chosen for recording if membrane seal resistance was greater than 1 GΩ and access resistance less than 40 MΩ. Voltage-clamp control and current measurements were made with an Axopatch 200B and digitized with a Digidata 1322 A interface and pClamp software (Molecular Devices). Cells were held at −100 mV between voltage steps with 200 ms steps in the activation protocol and 1 s conditioning steps immediately followed by 100 ms test step to +20 mV for the steady-state inactivation protocol. Extracellular fluid was delivered to the cells through a SF-77B perfusion system (Warner Instruments) at a rate of 1 ml/min. All experiments were performed at 22 °C.HEK293t cells were cultured in Dulbecco’s modified Eagle’s medium and 10% heat-inactivated fetal bovine serum (Invitrogen) containing penicillin and streptomycinAplysia californica were obtained from the National Resource for Aplysia at the University of Miami. Aplysia pleural-pedal ganglia were digested in dispase II, desheathed, and ventral-caudal cluster pleural sensory neurons (SN) were individually isolated and transferred to poly-L lysine coated, glass bottomed culture dishes in a culture media containing 10% hemolymph and 90% isotonic L-15 media. With synaptic pairs, postsynaptic LFS siphon motor neurons were isolated from the abdominal ganglia based on position and morphology, and placed next to a single SN in a culture media containing 50% hemolymph and 50% isotonic L-15. After 24 h the sensory neurons were injected and left for 48 h before synapse measurements. Sharp glass microelectrodes ~15 MΩ in resistance when backfilled with 2 M potassium acetate were used for recording membrane potentials and to evoke action potentials. PSPs were evoked with single action potentials in the SN with 50 ms pulses with the motor neuron held at −80 mV. PSP amplitude was measured from peak amplitude at synapses <20 mV and initial PSP rise-rates for larger PSPs to avoid amplitude errors from non-synaptic voltage-dependent currents . Electrodes were controlled with Sutter MP-225 micromanipulators and potentials amplified with an Axoclamp 900 A and digitized at 10 kHz with a Digidata 1440. The amount of current required to reach action potential threshold with a 50 ms pulse from a holding potential of −80 mV was used to measure membrane excitability.21, and injected at a concentration of 200 ng/μL. PP2 (Sigma-Aldrich) was dissolved in DMSO at a final bath concentration of 0.1%, applied to the bath at a 10-fold concentration twenty minutes prior to the experiment. The 5HT1A agonist (±)-8-Hydroxy-2-dipropylaminotetralin hydrobromide (8-OH-DPAT) and dopamine (Sigma-Aldrich) were applied to the bath as a bolus with a pipette.Nuclear injections of pNEX expression vectors occurred after 24 h in culture and the neurons were then recorded/imaged 48 h after that, unless stated otherwise. Nuclear injections of DNA were through custom fabricated micropipettes using backing pressure regulated with a WPI PV820 picopump. DNA injections did not include fast green to avoid artifacts from spectral bleed through from the fast green into the RFP channel during fluorescence imaging. The recombinant 5HT1A and eGFP-5HT1A receptor sequences in pNEX3 vectors used were as described ino, where Fo was the average fluorescence values measured over the ten frames preceding the frame before evoking an action potential.Fluorescence images were acquired on a Zeiss Axio Observer D1 through a EC Plan Neofluar 1.3NA 40 × oil coupled objective with a Photometrics QuantEM:512SC EM CCD camera. Standard Zeiss RFP filter sets (RFP 20) were used to image RFP intensity at maximum lamp intensity, constant exposure rate, CCD gain set at 20 for soma imaging and 200 for neurite imaging. RFP imaging was always done prior to Fluo-4 loading. For measuring RFP intensity in neurites, a circular ROI with a diameter equal to the width of the neurite was made at approximately 100 μm and 200 μm from the center of the soma. The mean background fluorescence measured at an adjacent ROI of the same size without any neurites was subtracted from the mean fluorescence intensity in the neurite. Fluo-4 pentapotassium salt (ThermoFisher Scientific) at 6 mM was backfilled in recording electrodes and iontophoretically injected into sensory neurons with hyperpolarizing pulses until sufficient intensity in the soma was reached . Fluo-4 imaging was conducted with a standard Zeiss GFP filter (GFP 38) set and a lamp intensity of 25% and 25 ms exposure times with 2 × 2 pixel binning to achieve frame rates of ~20 Hz with minimal bleaching during imaging. Three single action potentials were evoked at 0.2 Hz with sharp microelectrodes before and 30 s to 1 min after agonist. Regions of interest were chosen at neurite branch points or varicosities with both an RFP signal and Fluo-4 fluorescence transient with an action potential. All calcium imaging experiments were done in the presence of 20 μm nifedipine (Sigma-Aldrich) added to the dish five minutes before the experiment. With the exception of Fig. All images were analyzed using ImageJ and electrophysiological data analyzed with Clampfit. Statistical analyses were performed with Graphpad Prism and SPSS. All tests α = 0.05 and all data passed Kolmogorov-Smirnov normality tests prior to statistical comparisons. All data are means ± the standard error of the mean.Supplementary information