Eastern Beringia is one of the few Western Arctic regions where full Holocene climate reconstructions are possible. However, most full Holocene reconstructions in Eastern Beringia are based either on pollen or midges, which show conflicting early Holocene summer temperature histories. This discrepancy precludes understanding the factors that drove past  climate change and calls for independent proxies to advance the debate. We present a ~13.6 ka summer temperature reconstruction in central Yukon, part of Eastern Beringia, using precipitation isotopes in syngenetic permafrost. The reconstruction shows that early Holocene summers were consistently warmer than the Holocene mean, as supported by midges, and a thermal maximum at ~7.6–6.6 ka BP. This maximum was followed by a ~6 ka cooling, and later abruptly reversed by industrial-era warming leading to a modern climate that is unprecedented in the Holocene context and exceeds the Holocene thermal maximum by +1.7 ± 0.7 °C. Traditional precipitation isotope archives  are fundamental to our knowledge of past climate but limited to glaciated locales. Here the authors show that pore ice in relict permafrost holds equal promise as a proxy and use it to provide insights on the Holocene summer climate history of northwestern Canada. This warming is driving a range of biotic and abiotic changes that directly impact northern communities and ecosystems3, as well as enhanced C fluxes to the atmosphere from thawing permafrost4 and ice loss from glaciers and ice caps5, which have implications for global climate and sea level, respectively. The broad-reaching effects of Arctic change highlight the need to advance our knowledge of the Arctic system.The Arctic has warmed faster than any other region this past century, owing to climate-cryosphere feedbacks6. Arctic proxy reconstructions for the Common Era 7 and Holocene (last ~11.7 ka)9 broadly agree that the late Holocene was characterised by a long cooling trend that ended with abrupt warming during the industrial era. Many proxy reconstructions agree that industrial-era warming was exceptional in overall rate, but not magnitude in the Holocene context11, while others suggest that industrial-era warming has been exceptional in both regards13. Differences can be explained by spatial heterogeneity in the climate field or proxy-related noise . Noise is inherent to all proxies, but recovery of the climate signal can be optimised through replication and a diverse, multi-proxy approach.Current knowledge of the Arctic system is informed by a sparse network of relatively short (~50–100 years) instrumental records. However, the Arctic is transitioning to a warmer state that appears to have no analogue in the historical record. Natural proxy archives (e.g. ice cores and lake sediments) offer an alternative source of data on past climate and environmental change extending back thousands of years, with analogues from the distant past that may be especially relevant for anticipating the form of a future, warmer Arctic9 showed that nearly all quantitative, summer-specific temperature reconstructions from Eastern Beringia with early Holocene coverage are based on two proxy types—pollen and midges (Chironomidae) in lake sediments. Regionally, pollen and midges agree there was a climate optimum at ~7–5 ka BP, followed by a long-term cooling. However, these proxies show large differences before 8 ka BP with pollen indicating that early Holocene summers were persistently colder than the Holocene mean , and midges showing that early Holocene summers were similar to the Holocene mean . This discrepancy confounds our understanding of the drivers of early Holocene climate change and highlights the need for independent proxies to resolve this debate.Eastern Beringia, the contiguous area of Yukon and Alaska that was ice free during the last glaciation, is one Arctic borderland where the Holocene climate history is still debated. A review by Kaufman et al.2H/1H (δDprecip) and 18O/16O (δ18Oprecip)—in ice core records have a long history of use as a temperature proxy16 and have played a crucial role in shaping our knowledge of Quaternary climates in glaciated locales. More recently, this concept has been applied to Pleistocene and Holocene ice wedges (snowmelt derived) in permafrost environments in parts of Siberia17 and Eastern Beringia20 to reconstruct winter δprecip (δDprecip or δ18Oprecip) and temperature. Relict pore ice in syngenetic (aggrading) permafrost also shows potential as a δprecip proxy19, although there are few pore ice studies with a paleoclimate focus, and only one example from Siberia21 where a Holocene δpore ice series was developed and used for qualitative (warmer vs. cooler) temperature inferences.Precipitation isotope ratios—18O relative to the initial pore waters22. Based on this record, we develop a full Holocene δDprecip (pore ice)-based summer ΔT reconstruction, and an abiotic perspective to advance our understanding of early Holocene climate in Eastern Beringia.In this study, we present the first full Holocene, quantitative temperature reconstruction from a sequence of relict pore ice in syngenetic permafrost from a soligenous peatland called DHP174  in central Yukon Fig. , part of−1 average) from 14.3 to 11.2 ka cal BP. Unit 2 overlaps with the Younger Dryas interval , when the climate of northern Alaska was much colder than today23. Finally, Unit 3  is primarily Sphagnum moss, reflecting an aggrading peatland phase. The peat sequence accumulated at a relatively stable rate of ~0.4 cm decade−1, with the exception of higher rates at the time of peatland inception  and a brief slowdown near the core top  during the period ~1000–200 cal yr BP.The DHP174 peat-sediment composite record (5.32 m composite depth) is the product of two core profiles that overlap stratigraphically and includes three lithostratigraphic units Fig.  that wer24. Rare ice lenses  and Unit 3 (n = 2) but were not sampled. Ice lenses represent active layer waters drawn to the freezing front by cryosuction24, a process that is common in fine-grained sediments and may explain their higher number in Unit 1. Pore ice and lens ice derive from potentially different active layer water and cannot be directly compared. In this study, we focus exclusively on the dominant form of ice—relict pore ice.Peat fibres were exceptionally well preserved throughout the profile . ALTmax is defined as the thickest active layer likely to occur in a given climatic period and is ~58 cm for the study site today . For the lower core profile, we consider all pore ice at or below a composite depth of 290 cm (~90 cm below the lower coring surface) to be relict pore ice. The deeper cut-off depth to relict pore ice in the lower core avoids pore ice that has been overprinted by modern waters following disturbance of the lower surface , the upper core being relatively depleted compared to the lower core in δD and δ18O   and are not unexpected for different sites across a landscape given the same source water. As we discuss below, relict pore ice is enriched in heavy isotopologues relative to the initial pore waters due a fractionation that occurs during the freezing process22. The overall enrichment (εice-water) depends on the rates of freezing and molecular diffusivity. For our site, the lower active layer is saturated in the late summer and, thus, the rate of freezing will be buffered by latent heat effects25. However, water table depth and total latent heat content are unlikely to be spatially homogenous due to variations in microtopography and slope, and therefore the rate of freezing and εice-water are also unlikely to be homogenous. Because the two core locations (~50 m apart) receive the same precipitation and runoff, spatial variability in εice-water is a probable explanation for the small offsets in δpore ice.The upper and lower relict pore ice records collectively span the last ~13.6 ka cal BP. The two records show small systematic differences in their mean δD and δ18O Fig. . There i8O) Fig.  The δD a2–103 year timescales .Regardless of origin, the offsets are systematic and the cores show coherent variations at 10les Fig. , which sets Fig. . This alrds Fig.  also access Fig.  nor concThe composite record covers the last ~13.6 ka with an average time-step of ~158 years between pore ice samples since 10.5 ka cal BP; however, from 13.6–10.5 ka cal BP, the average time-step is relatively coarse (~1020 years between samples) due to low accumulation rates just prior to the peatland initiation.18O) measurements of relict pore ice plot tightly on the Local Meteoric Water Line  that once formed at the bottom of a paleo-active layer. First ice is enriched in heavy isotopes relative to the initial pore waterspore water and δ18Opore water estimates for the uppermost 3 cm of DHP174-13 relict pore ice  are −161.5 and −20.2‰, respectively, with a mean dexcess value of +0.1‰. Compared to local meteoric waters from Mayo, our δDpore water estimate is enriched relative to mean annual δDprecip and within range of thaw season (May–September) δDprecip values  and ca. modern pore ice (last ~3 decades) are unequal, which may impact how closely they agree. Mayo δDprecip data generally follow expectations of more depleted values during cold winter months and enriched values during warm summer months , which are likely attributable to the limited replication in the Mayo record. The proposed seasonality of relict pore ice can also be corroborated by comparing δpore ice-based temperature estimates (Methods) with the local climatology as constrained by climate data from the Ogilvie River station (Supplementary Note σ) based on δDpore ice  based on δ18Opore ice  is high 27. Conversely, as the active layer thaws in the summer, precipitation percolates to greater depths and will drain slowly due to lower Ks in the deeper active layer (0.5–5 m day−1 below 20 cm)27. This hydrological style favours greater representation of warm-season precipitation in the lower active layer, where pore waters eventually freeze and become relict following permafrost aggradation.A warm-season seasonality is also expected for climatological and hydrological reasons. Roughly 65% of the annual precipitation budget is delivered during the thaw season, the bulk of which is rain Fig. . Secondlpore ice variability (on average 0.4‰ for δD and 0.1‰ for δ18O) in comparison to the large inter-annual (weather-related) variability in δprecip that is evident in local GNIP records for most months , which dictates that up to ~20–30 years of mostly warm-season precipitation can mix in the lower active layer before the permafrost table aggrades 1 cm, thereby trapping pore ice situated at the former permafrost table as constrained by ALTmax. While ALTmax will not be reached every thaw season (Supplementary Note max) during prior, cooler summers. Thus, natural averaging of multi-year pore ice in the active layer ensures a climatic timescale integration (depending on the aggradation rate) of δprecip and representation of cool and warm summers in any given sample of δpore ice, which explains the lack of extreme inter-sample δpore ice variability noted above.Finally, we note that the composite record of pore ice Fig.  is charapore ice records were corrected for changes in δseawater  record  during the late pre-industrial era.Over Holocene and longer timescales, the isotopic composition of seawater is variable. The composite δord Fig.  shows a 18Opore ice (seawater corrected) record , dexcess values >4‰ are common during the cold-season months (October–April), and so the dexcess change in our pore ice record could be interpreted as a change in the precipitation seasonality (e.g. ratio of warm-to-cold-season precipitation) integrated in pore ice; however, this interpretation would also require a constant LMWL, which the co-isotope results  defined by early Holocene relict pore ice  to the contemporary LWML defined by modern GNIP data, but which aptly describes late Holocene relict pore ice younger than 5  ka BP  is influenced by the mean evaporative boundary conditions at the moisture source28. Given the similar slopes but different intercepts of the paleo vs. modern LMWLs, we conclude that a change in evaporative boundary conditions at the moisture source is the most likely explanation for the Holocene dexcess transition.The δord Fig.  documentord Fig. , which tlts Fig.  show is  BP Fig. . Continedexcess parameter reflects non-equilibrium partitioning of HDO relative to the slower moving H218O isotopologue in the evaporate leaving the ocean. dexcess is sensitive to factors that influence the evaporation rate including sea surface temperature (SST) and relative humidity (RH)28, which has made it a useful proxy in ice core studies for reconstructing changes that are far removed from the ice core site14. The North Pacific is the primary moisture source for central Yukon today19. Assuming a stable moisture source through the Holocene, the observed dexcess trend could reflect a cooling in North Pacific SST and/or rise in RH in the moisture source region. SST proxies encompassing a large area of the North Pacific broadly agree that SSTs peaked around ~10 ka and cooled by ~1–2 K by the mid-Holocene30. However, spatial coherence diminishes in the late Holocene with some SST reconstructions, indicating warming and others cooling. The balance of paleo-SST evidence is inconclusive, but hints at a possible and spatially complex relation between dexcess and SST.The dexcess trend could indicate a geographic shift in the moisture source region with potentially different mean SST or RH at the different localities. The initial timing of the dexcess trend is coincident with the final collapse of the Laurentide ice sheet (LIS) from ~11–7 ka BP31. The LIS strongly impacted atmospheric circulation and climate in western Eurasia32. Likewise, climate modelling studies suggest that the collapse of the LIS had a warming effect on E. Beringia33, although the dynamical response of summertime atmospheric circulation patterns in the North Pacific sector to LIS collapse is unknown.Alternatively, the precip boundary conditions to another. Although the physical mechanisms behind this transition are not well known, further interrogation of possible changes—such as modified SST or RH at a constant moisture source, or a geographic change in moisture source —will benefit from greater replication of the paleo-SST field and isotope-enabled GCM  experiments.The co-isotope results indicate a transition from one set of δprecip boundary conditions, δDprecip and δ18Oprecip are both temperature-sensitive proxies  were applied to the δDpore ice and δ18Opore ice records, they would yield different reconstructions given their contrasting trends .Under modern δnds Fig. —and cleaT reconstruction  with a mean ΔT of +0.1 °C. Local summer ΔT reconstructions from pollen and midges show major differences during this interval, with pollen indicating cool ΔT values from –2.2 to –1.2 °C, and midges indicating warm values from +0.1 to +0.4 °C , 8.8 ka cal BP (+0.4 °C), 7.6 ka cal BP (+0.4 °C) and 6.8–6.6 ka cal BP (+0.3 °C). Individual ΔT anomalies should not be over-interpreted; however, it is notable that the +0.4 °C anomaly at 8.8  ka cal BP coincides with a deep thaw unconformity preserved in permafrost near Mayo, which is dated to 8.87 ± 0.2 ka BP and has been linked to a brief period of exceptionally warm conditions34. We also note that the later warm interval between 7.6 and 6.6 ka cal BP coincides with a period of Arctic treeline expansion in the Mackenzie Delta region (~400 km N.E. of DHP174) that began ~8 ka BP35. The 7.6–6.6 ka cal BP warm interval also matches a thermal maximum in the extratropical composite , but slightly warmer summers (+0.55 °C) from 6.0 to 5.5 ka BP  are relatively small, and to some extent are likely explained by the unequal spatial representation of the different records. Additional pore ice records across the region may improve inter-proxy coherence.From 9.6 to 6.6 ka cal BP, our reconstruction shows a sustained warming with Δ °C Fig. . Local mite Fig. . The E. ite Fig. . The reg BP Fig. . Similar−1 (p = 6.82 × 10–5)  between 6.6 and 1.2 ka BP. Regional pollen and midge composites also show cooling trends through this interval, but at lesser rates of change  Fig. . Promine–5) Fig. . The ext36, and is not supported by the multi-proxy composites from 30 to 90°N , the late pre-industrial era is not well resolved in our pore ice record; therefore, our reconstruction should not be used for detailed estimates of temperature change during this interval.From ~1200 to 150 cal yr BP our reconstruction shows a gradual warming trend. A similar warming trend over the last ~1–2 ka is observed in the E. Beringia all-temperature composite Fig. . However0°N Fig.  and 60 t0°N Fig. , which sT anomalies during the early-middle Holocene thermal maximum  is +1.9 ± 0.7 °C warmer than the Holocene mean. The last ~100 years of our record is especially well resolved  due to the finer pore ice sampling resolution through this interval, and the historic cryptotephra (1912 CE Novarupta-Katmai at 10 cm depth) and post-bomb 14C dating  that constrain this part of the chronology  pollen-based temperature reconstructions suggest the last decade was ~1.2 °C warmer than the Holocene mean13. Retreating glaciers on Baffin Island in the Canadian Arctic have exposed rooted plant fossils with non-finite 14C ages, which indicates that recent climate warming at that locality is unprecedented since the last interglacial6. While these select examples do not resolve the debate over whether today’s climate is exceptional everywhere in the Holocene context, they do support the idea that the magnitude of early Holocene warming has varied spatially. At our site in easternmost Beringia, the present-day summer climate does appear to be exceptional in the last 13.6 ka cal BP, and our reconstruction suggests that summer temperatures in recent decades have consistently exceeded the Holocene Thermal Maximum since about the mid-20th century.Finally, our reconstruction shows accelerated warming since ~150 cal yr BP, culminating in the warmest summer climate at present, which  we estimate is 2.1 ± 0.7 °C warmer than the Holocene average or 1.7 ± 0.7 °C warmer than the peak Δmum Fig. . The avey Tables , S2. Our 37 Fig. . Similar 37 Fig. , and tem33. However, other factors likely had a moderating effect. For example, the flooding of the Bering Strait (as late as 11 ka BP38) and development of thaw lakes in Yukon and Alaska in the early–middle Holocene probably had a regional cooling effect on summer climate33, while the LIS collapse and increases in shrub and boreal taxa likely had a warming effect39. The integration of all relevant forcings and feedbacks likely explains some of the more complex variabilities in early Holocene climate that diverge from a simple insolation-based interpretation. Finally, the industrial-era warming in our record (~2 °C warming over the last century) is coeval with the rapid increase in global pCO2 41, which likely accounts for at least part of the 20th century warming trend in our record.Variability in Holocene summer climate in this region owes to multiple factors. The centroid of the July–August insolation curves and subsequent decline is closely aligned with the low-frequency trend in our reconstruction Fig. , and impCO2 Fig. , which i3. Cryostratigraphic evidence for regional failure of ice-rich permafrost in E. Beringia is linked to past hypsithermals, including thermokarst deposits in Alaska and Yukon dating to Marine Isotope Stage 5e42, and thaw unconformities and thaw lake initiation dating to the early Holocene in parts of Yukon and the Mackenzie Delta region45. However, the warming threshold for such a widespread permafrost response is not well known. Our finding that today’s summer climate (last ~30 years) exceeds the early–middle Holocene thermal maximum by +1.7 ± 0.7 °C implies that the threshold for ice-rich permafrost stability in this region has already been crossed. This finding is consistent with recent evidence of climate-driven acceleration of thaw slump activity46 and ice wedge network degradation47 in the Mackenzie Delta region and Western Arctic more broadly, which have been linked to climate warming. Continued warming and permafrost degradation in this region threatens to expose vast pools of frozen organic matter in near-surface permafrost to microbial activity and, if released to the global atmosphere as CO2 or CH4, has the potential to amplify future warming trends4.The rate and magnitude of recent climate warming has implications for the stability of ice-rich permafrost in E. Beringia, which is both widespread and sensitive to climate warmingSphagnum spp. moss, and includes sparse forb and lichen groundcover, and an open canopy of Picea mariana trees. DHP174 is a soligenous peatland, sustained by precipitation and seasonal runoff from a south-flanking hillslope. There is ~15–20 m of relief across the ~100–150 m peatland surface, measured from the bottom of the hillslope to the northern edge of the peatland48. The slope ensures continuous drainage of surface and active layer runoff throughout the thaw season, and well-drained conditions in the uppermost 20–30 cm of peat. Saturated conditions during the summer occur below 20–30 cm depth and are maintained by elevated summer precipitation totals  that are typical below 30 cm depth in northern peat plateaus27. The DHP174 peatland sits on an alluvial terrace in a valley that drains into Engineer Creek  is from the ‘undisturbed’ upper surface, and the second core profile  was collected from an ‘excavated’ lower surface  to core bottom (382 cm composite depth); the only exception being DHP174-13 core depths 16–76 cm, which were analysed at near 1 cm resolution to allow for a more detailed analysis of pore ice in the active layer and across the active layer–permafrost interface. For the DHP174-13L core, every tenth centimetre increment of pore ice was analysed for water isotopes starting at a depth of 30 cm below the lower coring surface  down to the basal sediments at 530 cm composite depth). In total, 104 pore ice samples were analysed for water isotope ratios, 43 of which are active layer or transient layer pore ice, and 71 of which are relict pore ice.18O ratios of the water were measured at the University of Alberta using a Picarro L2130-i analyser. Unknowns and standards were injected eight times, and mean raw values were calculated from the last four injections. Raw isotope ratios were corrected for inter-sample memory effects that are common to cavity ring-down spectroscopy systems50. We used certified water reference standards (USGS-45 and USGS-46) to normalise raw isotope ratios to the Vienna Standard Mean Ocean Water-Standard Light Antarctic Precipitation (VSMOW-SLAP) scale. Analytical error is 0.6‰ for δD and 0.1‰ for δ18O based on routine analysis of an internal deionised water standard.The frozen samples were thawed at ~4 °C in plastic bags and the liquid was filtered into 2 mL autosampler vials. δD and δ14C) dates and cryptotephra isochrons (see Davies51 for full details of the composite chronology). Depths are reported as core composite depths (ccd). The Bayesian age-depth model  were used for 14C dating. Samples were pre-treated at the University of Alberta following standard procedures54, and analysed for 14C content at the W. M. Keck Carbon Cycle AMS Laboratory . Two secondary standards were pre-treated concurrently  and the 14C results were within expected ranges. The unknown 14C dates were calibrated as appropriate using Bomb13NH155 and IntCal1356 calibration curves with Oxcal v4.3.257.Where possible, 58. Three samples geochemically correlated with known eruptions using major element EPMA glass analyses  were included in the composite age-depth model to improve model accuracy. Katmai-Novarupta (10 ccd) is a historic eruption with an observed occurrence date of 1912 CE. The Aniakchak CFE II (116 ccd) and Hayes set H unit F2 (133 ccd) eruptions are widely reported in Alaska and beyond, and have well-constrained Bayesian age estimates59.Glass shard concentration profiles for cryptotephra were produced at 1 cm resolution using standard methodsmax in Supplementary Note max of 58 cm through time. Although ALTmax potentially did vary throughout the Holocene due to changes in summer climate, it likely remained within 3–6 cm of the modern ALTmax value and does not significantly influence our pore ice chronology or conclusions  were given a weight of zero in the calibration to avoid undue bias on the regression lines. By comparison with monthly GNIP data from Whitehorse . To isolate local climate signals in δprecip (pore ice), which are unrelated to changes in seawater, the composite pore ice record was corrected for long-term δseawater changes following the general equation .Two types of temperature estimates were made based on the pore ice record—mean temperatures for the modern era Fig.  and a fuT) Fig. . The firT) were reconstructed for the full Holocene based on δDpore ice anomalies  and the δDprecip–temperature slope (m). Since εice-water is assumed to be a constant (Supplementary Note pore ice and ΔδDprecip are equal. The formula used for estimating ΔT is: ΔT = [ΔδDpore ice ± 1σ]/[m ± 1σ].Temperature anomalies (ΔSupplementary InformationSupplementary Data 1


Fibrocytes were characterized in whole blood by flow cytometry, and in culprit site thrombi and myocardium by immunofluorescence. To investigate mechanisms of fibrocyte activation, isolated NETs were used to induce fibrocyte responses in vitro. Enzymatic infarct size was assessed using creatine-phosphokinase isoform MB area under the curve. Left ventricular function was measured by transthoracic echocardiography. NET surrogate markers were increased at the culprit site compared to the femoral site and were positively correlated with infarct size and left ventricular dysfunction at follow-up. In vitro, NETs promoted fibrocyte differentiation from monocytes and induced fibrocyte activation. Highly activated fibrocytes accumulated at the culprit site and in the infarct transition zone. Our data suggest that NETs might be important mediators of fibrotic remodeling after STEMI, possibly by stimulating fibrocytes.Leukocyte-mediated inflammation is central in atherothrombosis and ST-segment elevation myocardial infarction (STEMI). Neutrophil extracellular traps (NETs) have been shown to enhance atherothrombosis and stimulate fibroblast function. We analyzed the effects of NETs on cardiac remodeling after STEMI. We measured double-stranded (ds)DNA and citrullinated histone H3 (citH3) as NET surrogate markers in human culprit site and femoral blood collected during primary percutaneous coronary intervention (The online version of this article (10.1007/s00395-019-0740-3) contains supplementary material, which is available to authorized users. ST-segment elevation myocardial infarction (STEMI) is among the leading causes of death and morbidity . MountinLong-term outcome after STEMI is significantly affected by adverse fibrotic remodeling, with fibroblasts being major determinants . FactorsWe queried whether NETs exerted a fibrotic stimulus in the setting of STEMI, and whether this stimulus occurred via fibrocytes, leading to adverse cardiac remodeling after STEMI.Detailed information on material is provided in Supplemental Table S1.n = 50) undergoing primary percutaneous intervention (pPCI) for a coronary TIMI flow of 0. Patients were included if they met the following criteria: (1) chest pain at the time of coronary angiography; (2) new ST-segment elevations of ≥ 2 mm on at least one chest lead or elevations of ≥ 1 mm on at least one limb lead within 20 min of angiography; and (3) suitable coronary anatomy for thrombus aspiration. All other inclusion and exclusion criteria, and general procedural considerations for use of a thrombectomy device, were applied as previously described  days after STEMI. Echocardiography at long-term follow-up (24 ± 8 months) was performed in 24 patients. Left ventricular ejection fraction (LVEF) was measured using biplane modified Simpson’s method. Myocardial contractility was assessed and scored for each segment as described : (1) nort test was used; otherwise, Wilcoxon signed-rank test was computed. For unpaired comparisons, unpaired Student’s t test was employed in case of normally distributed data; otherwise, Mann–Whitney U test was used. For comparison of three groups, one-way analysis of variance with Dunn’s multiple post hoc comparison was employed. Correlations between parameters were assessed using Spearman’s rank correlation. To correct for multiple testing, the Bonferroni–Holm method was used. Statistical analyses were performed using IBM SPSS 21.0. Figures were generated using GraphPad Prism 5. Data are depicted as scatter plots; lines in graphs indicate mean ± standard error of the mean, in case of normally distributed data; otherwise, median and IQR are given.Distribution of data was analyzed using the Kolmogorov–Smirnov test and histograms (data not shown). Normally distributed numerical data are presented as mean ± standard deviation (SD); otherwise, median and interquartile range (IQR) are provided. To compare paired and normally distributed data, paired Student Characteristics of patients with STEMI and TIMI flow of 0 at pPCI are shown in Table n = 48, culprit site dsDNA 529 [429–740] vs. femoral dsDNA 404 [349–563] ng/ml, p < 0.0001, Fig. n = 48, culprit site 332 [123–810] vs. femoral 235 [113–434] ng/ml, p < 0.01, Fig. n = 21, 291 [252–327] ng/ml, p < 0.0001, Fig. n = 21, 192 [150–399] ng/ml, p = ns, Fig. In a first series of experiments, we measured concentrations of dsDNA and citH3 as surrogate markers of NET burden. Consistent with our previous findings , dsDNA wn = 46, culprit site 0.12 [0.01–0.83] vs. femoral 0.00 [0.00–0.08] pg/ml, p < 0.001, Supplemental Fig. S5a). At 72 h after pPCI, EV-TF activity was not different compared with femoral site values . EV-TF activity at the culprit site was positively correlated with dsDNA , but not with citH3 .Tissue factor (TF) is important for the primary activation of the coagulation protease cascade and is thus paramount for hemostasis and thrombosis , 19. RecMonocytes have been shown to be precursors of fibrocytes . Upon inIn a next step, we stimulated cultivated fibrocytes with isolated NETs in vitro for 6 h. NETs induced an upregulation of collagen-I Fig. a, BMPRII+CD34+collagen-I+ cells in particulate coronary thrombi, confirming the presence of fibrocytes .We analyzed fibrocyte frequency and function 72 h after pPCI. Compared to baseline, we observed a decline of the number of circulating fibrocytes at 72 h Fig. a. This wn = 47, rs = 0.321, p < 0.05). Furthermore, a positive correlation was found between citH3 and CD34 expression . A positive correlation was observed between dsDNA at the culprit site and BMPRII expression of culprit site fibrocytes .Since NETs were reported to promote fibroblast function , and basn = 4, 2.7 ± 0.6% of cells; Fig. n = 3, 0.9 ± 0.2%, p = 0.007 for STEMI vs. healthy; Supplemental Fig. S7).Fibrocytes were detected mostly in the transition zone between viable myocardium and scar of patients dying from acute STEMI  being an important ligand of CD11b . ICAM-1 In a subset of patients, we obtained transthoracic echocardiographic measurements at long-term follow-up. dsDNA at the culprit site at the time of pPCI was positively correlated with WMSI at follow-up Fig. a. FibrocThe conceptual framework is summarized in Fig. dsDNA was highly increased at the culprit site compared with the femoral site, confirming previous data , 60. FurIt was previously shown that fibroblasts are stimulated by NETs  and we fIn parallel to NETs, fibrocytes accumulate at the culprit site and are present in coronary thrombi. Increased expression of collagen-I on culprit site fibrocytes compared to the femoral site suggests that they become locally activated and might contribute to tissue remodeling via deposition of extracellular matrix. Collagen-I expression on peripheral fibrocytes 72 h after STEMI was upregulated and suggests a sustained systemic activation.Hematopoietic stem cells have been shown to migrate to myocardial tissue after injury . CD34 isCD11b is expressed on fibrocytes , 50 and The frequency of peripheral fibrocytes was lower 72 h after STEMI compared to baseline. We thought that fibrocytes might migrate to the ischemic myocardium. As expected, we detected fibrocytes within the myocardium of patients with STEMI. Our findings point towards homing of fibrocytes to the myocardium during and after STEMI. These observations are corroborated by a mouse model of ischemia/reperfusion cardiomyopathy showing an increase of fibrocytes in the heart after ischemic challenge . A studySeveral pathways have been implicated in atherosclerosis, among them bone morphogenic protein (BMP) signaling . BMP recSoluble NET markers were shown to be associated with adverse cardiac events . The preCurrently, limited evidence exists on cardioprotective strategies for reducing myocardial damage in MI . Thus, tThis is a translational, hypothesis-generating study. Given the observational nature of our findings, causality cannot be inferred. citH3 has been extensively used for the identification of NETs both on the tissue level , 57, 69 Measurement of DNase as the natural counter-regulatory mechanism of nucleosome generation in STEMI was not performed because two different DNase types are relevant in vascular biology and would have been needed to be addressed . In thisSupplementary material 1 (DOCX 923 kb)Below is the link to the electronic supplementary material.


Introduction: Excessive co-contraction interferes with smooth joint movement. One mechanism is the failure of reciprocal inhibition against antagonists during joint movement. Reciprocal inhibition has been investigated using joint torque as an index of intensity during co-contraction. However, contraction intensity as an index of co-contraction intensity has not been investigated. In this study, we aimed to evaluate the influence of changes in contraction intensity during co-contraction on reciprocal inhibition.Methods: We established eight stimulus conditions in 20 healthy adult males to investigate the influence of changes in contraction intensity during co-contraction on reciprocal inhibition. These stimulus conditions comprised a conditioning stimulus-test stimulation interval  of -2, 0, 1, 2, 3, 4, or 5 ms plus a test stimulus without a conditioning stimulus (single). Co-contraction of the tibialis anterior and soleus muscles at the same as contraction intensity was examined at rest and at 5, 15, and 30% maximal voluntary contraction (MVC).Results: At 5 and 15% MVC in the co-contraction task, the H-reflex amplitude was significantly decreased compared with single stimulation at a 2-ms C–T interval. At 30% MVC, there was no significant difference compared with single stimulation at a 2-ms C–T interval. At a 5-ms C–T interval, the H-reflex amplitude at 30% MVC was significantly reduced compared with that at rest.Discussion: The findings indicated that during co-contraction, reciprocal Ia inhibition worked at 5 and 15% MVC. Contrary inhibition of reciprocal Ia inhibition did not apparently work at 30% MVC, and presynaptic inhibition (D1 inhibition) might work. In many upper motor neuron disorders including spastic diseases, cerebellar ataxia, Parkinson’s disease, and spinal cord injury, when movement of only an agonist is required , a collapse of the reciprocal inhibition mechanism against the antagonist causes excessive co-contraction. Thus, reciprocal inhibition does not work on the antagonist, impairing smooth joint movement . Such exA previous study on reciprocal inhibition during co-contraction of the tibialis anterior (TA) and soleus (Sol) muscles in healthy men reported that the amount of reciprocal Ia inhibition was further decreased during co-contraction than at rest . In thatThis study thus aimed to clarify the influence of changes in contraction intensity during co-contraction on reciprocal inhibition. Other researchers e.g.,  have repTwenty healthy adult males  provided written informed consent to participate in this study. The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee at Niigata University of Health and Welfare.The right lower limb position was measured at the hip (90°), knee 120°), and ankle (90°) joints. The ankle was immobilized using a foot plate   of the surface electromyogram was set to 20 mm. The electrodes were placed on the TA and Sol muscles according to SENIAM . The groMuscles were stimulated for 1 ms (rectangular wave) using a SEN-8203 electrical stimulation device  via a SS-104J isolator (Nihon Kohden). The tibial nerve was selectively stimulated in a monopolar fashion to induce the Sol H-reflex and M waves. The anode and cathode were located on the upper patella and the popliteal area, respectively, for the test stimulus. M waves were induced in the TA using bipolar stimulation, and a conditioning stimulus was applied along the deep peroneal nerve below the fibula head .Reciprocal inhibition was measured as described previously . A test The contraction intensities of TA and Sol were determined by measuring the maximal voluntary contraction (MVC). MVC was performed for plantar- and dorsiflexion with the thighs and feet fixed as shown in Figure The experimental procedure is shown in Figure Post hoc Tukey–Kramer multiple comparison tests were then performed. The seven stimulation conditions were compared with the single condition using paired t-tests with the Bonferroni correction for multiple comparisons. Co-contraction tasks under each stimulation condition were compared using two-way repeated-measures ANOVA with the factors, co-contraction task, and stimulation condition, followed by post hoc Tukey–Kramer multiple comparison tests. Statistical significance was set at p < 0.05.Peak-to-peak Sol H-reflex amplitudes were calculated as mean ± standard error (SE). The reciprocal inhibition of each co-contraction task was calculated as a percentage by dividing the Sol H-reflex amplitude by the Mmax amplitude ([Sol H-reflex amplitude/Mmax amplitude] × 100). To compare the amount of reciprocal inhibition between simultaneous contraction tasks under the seven stimulation conditions (excluding the single condition), the H-reflex amplitude for the conditioning stimulus was divided by the H-reflex amplitude of only the test stimulus and expressed as a percentage ([Conditioned H-reflex amplitude/Test H-reflex amplitude] × 100). Values are expressed as mean ± standard deviation. Iterative values were generated using two-way repeated-measures analysis of variance of the factors, co-contraction task, and stimulation conditions. F = 36.271, p < 0.001, partial η2 = 0.656], but not of the co-contraction task . In addition, the stimulation condition and the co-contraction task interacted significantly . Analyses of the single Sol H-reflex amplitudes during each co-contraction task and multiple comparisons did not identify any significant differences. These findings confirmed that changes in the Sol H-reflex amplitude in response to conditioning stimuli do not depend on the test stimulus intensity.In the two-way repeated measures ANOVA of the co-contraction task and stimulation conditions, there was a main effect of the stimulus condition  of each stimulation condition but not that of the co-contraction task . Additionally, the stimulation condition and co-contraction task interacted significantly . Multiple comparison tests, followed by post hoc analyses, did not identify any significant differences between co-contraction tasks at C–T intervals of -2, 0, 1, or 2 ms. In the co-contraction task at 30% MVC at a C–T interval of 4 ms, the H-reflex amplitude was significantly reduced compared with that at rest (p = 0.009) and at 5% MVC (p = 0.014). In the co-contraction task at 30% MVC with a C–T interval of 5 ms, the H-reflex amplitude was significantly reduced compared with that at rest (p < 0.001) as well as that at 5% (p < 0.001) and 15% MVC (p = 0.045).Figure The main findings of the present study are that reciprocal Ia inhibition occurred at 5 and 15% MVC due to changes in contraction intensity during co-contraction at the same intensity between the TA and Sol muscles. In addition, we demonstrated that the amount of reciprocal Ia inhibition during co-contraction of 5 and 15% MVC was the same as that at rest. Reciprocal Ia inhibition was not evident at 30% MVC, suggesting D1 inhibition.Our findings at rest were similar to those of a previous study , which sDuring co-contraction at 5 and 15% MVC, the Sol H-reflex amplitude was significantly decreased at a C–T interval of 2 ms compared with the single condition. These findings are similar to our hypothesis, as reciprocal inhibition differed from previous findings, and the extents of reciprocal Ia inhibition at 5 and 15% MVC were the same as those at rest. Previous studies have investigated changes in the amounts of reciprocal Ia inhibition during co-contraction at the same joint torque during plantar- and dorsiflexion . By contAt 30% MVC for the single condition and C–T intervals at 2 ms, the Sol H-reflex amplitude did not differ significantly. This result suggests that reciprocal Ia inhibition decreases in the Sol muscle depending on its contraction intensity. Excessive simultaneous contraction intensity also decreases joint movement . The intThe H-reflex amplitude was significantly decreased at 30% MVC compared with that at rest at C–T intervals of 4 and 5 ms. Previous studies have reported the involvement of D1 inhibition in the inhibition mechanism at C–T intervals of 5 ms or longer . D1 inhiOne limitation of this investigation is that it studied reciprocal inhibition at a middle latency of 3 to 5-ms C–T intervals. In reciprocal inhibition, short latency is reciprocal Ia inhibition (2 ms) and long latency is D1 inhibition (5–80 ms) . The repReciprocal Ia inhibition under changes in contraction intensity during co-contraction is yet to be fully elucidated. The present study confirmed reciprocal Ia inhibition at TA and Sol co-contraction at 5 and 15% MVC at the same contraction intensity. During co-contraction at 5 and 15% MVC, antagonist inhibition may occur during joint movement. However, co-contraction at 30% MVC decreased reciprocal Ia inhibition, suggesting that joint fixation could be affected. The influence of changes in co-contraction strength on reciprocal inhibition at intermediate latency requires further investigation, and suppression during co-contraction should be studied by combining contraction intensity with joint torque. Future studies should also investigate the influence of changes in co-contraction intensity on the reciprocal inhibition of middle latency, as well as reciprocal inhibition during co-contraction, by combining contraction intensity and joint torque.RH designed the study and wrote the initial draft of the manuscript. ME, SK, and HO contributed to analysis and interpretation of data and assisted in the preparation of the manuscript. All other authors have contributed to data collection and interpretation and have critically reviewed the manuscript. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thermotoga maritima to date, was investigated by docking simulations. XOS with backbone lengths of two to five (X2–X5) were docked into the active-site cleft of TmxB by AutoDock The modeled complex structures provided a series of snapshots of the interactions between XOS and TmxB. Changes in binding energy with the length of the XOS backbone indicated the existence of four effective subsites in TmxB. The interaction patterns at subsites −2 to +1 in TmxB were conserved among GH10 xylanases whereas those at distal aglycone subsite +2, consisting of the hydrogen bond network, was unique for TmxB. This work helps in obtaining an in-depth understanding of the substrate-binding property of TmxB and provides a basis for rational design of mutants with desired product profiles.Glycoside hydrolase family 10 (GH10) xylanases are responsible for enzymatic cleavage of the internal glycosidic linkages of the xylan backbone, to generate xylooligosaccharides (XOS) and xyloses. The topologies of active-site cleft determine the substrate preferences and product profiles of xylanases. In this study, positional bindings and substrate interactions of TmxB, one of the most thermostable xylanases characterized from  In techanism . The catechanism . Amino aechanism . The subechanism . The catechanism . Highly echanism ,16. In gechanism ,18. Although the GH10 xylanases share a similar overall fold and catalytic mechanisms, the polymorphisms of subsites, in particular of the distal regions of active-site cleft, result in the different substrate binding preferences . The subThermotoga maritima MSB8, is one of the most thermostable GH10 xylanases characterized to date [In this study, we showed that snapshots of GH10 xylanases and XOS interaction can be rapidly obtained by in silico study. TmxB, a GH10 xylanase produced by  to date ,20,21. TThe crystal structure of TmxB has been determined (PDB entry: 1VBU) . Basing Although a bulky grid box, including the entire active-site cleft (subsites −3 to +3), served as the platform for X2 docking, the resulting docking poses indicated that the X2 molecule was only bound to the −2 to −1 subsites a. The loA −2 to +1 positional binding was observed when X3 was docked into the active-site cleft of TmxB a and exhThe docking complex of TmxB-X4 with the lowest docking energy  indicated that X4 was bound to the −2 to +2 subsites a. The coDocking simulation indicated that the X5 molecule occupied the −2 to +3 subsites, which perfectly fitted with the shape of the active-site cleft in TmxB a. The doThe polymorphisms of subsites in the active-site cleft result in different substrate preferences and product profiles of individual GH10 xylanases . Amino aGeobacillus stearothermophilus) in complex with X3  (PDB entries 3MUA and 3MSG) (G. stearothermophilus) (E159Q) in complex with X5 (PDB entry: 4PUD), although the amino acid composition at the aglycone subsites were significantly variable between TmxB and XT6  , only thnd 3MSG) b. The pond 3MSG) . X4 was nd 3MSG) c. The sund 3MSG) c. A bind and XT6 d. Arg263Structural analysis of the docking complexes revealed additional details of the interaction between XOS and TmxB. In all of the docking structures, the conformations of −1 xylose residues were highly uniform, and no obvious differences were observed for the −1 subsite interactions between TmxB and other GH10 xylanases . The −2 The docking structures indicated that the xylose residues at the more distal aglycone subsite +2 and +3 interacted with the polar amino acids (Arg263 and Ser197) through the hydrogen bond network. Such an interaction pattern was different from the predominantly hydrophobic stacking interactions between GH10 xylanases and substrates at aglycone subsites . For exaCbXyn10C from Caldicellulosiruptor bescii [The overall XOS positional binding and corresponding cleavage pattern in TmxB are shown in r bescii .The amino acid sequences and the three-dimensional structures of TmxB and other representatives of GH10 xylanases were downloaded from the GenBank protein database  and PDB The crystal structure of TmxB (PDB entry: 1VBU) was used as the receptor for the docking simulations. Waters in the structure were removed, and the structure was minimized using UCSF  Chimera . The NE1Three-dimensional structures of XOS with backbone lengths of 2 to 5 were used as ligands for docking. Their structures were obtained from deposited structure complexes of GH10 xylanase in PDB. Specifically, X5, X4, X3, and X2 were from PDB entries 1E5N, 1US2, 4L4P, and 3NJ3, respectively. Enzyme-ligand docking simulations were conducted using the AutoDock Tools 1.5.6 platform (ADT) . A grid The amino acid sequence and structure alignments showed that the aglycone subsites in TmxB were nonconserved. Molecular docking revealed the roles of these subsite amino acids in positional binding and xylose residue interactions. Theoretical complexes obtained by docking of XOS (X2–X5) into the active-site cleft of TmxB showed that the −2 to +2 subsites in TmxB played a critical role in substrate binding. The +2 subsites anchored the xylose residue through a hydrogen bond network rather than the general hydrophobic stacking interaction at the aglycone subsites in GH10 xylanases. On the basis of substrate interactions observed in this in silico study, the consequences of mutations at the subsites could be predicted, which helps in alerting the character of TmxB though rational design. In addition, our results indicated the effectiveness of docking simulation in investigating the interaction between glycoside hydrolase and short oligosaccharides.


Triple 3 showed higher reduction than Triple 2 in IgE-binding (5.5 fold) and in histamine release (15.7 fold) compared to wild type Equ c 1. Mutations designed on the putative IgE epitope region and monomer-monomer interface of Equ c 1 resulted in decreased dimerization, a lower IgE-binding capacity and a reduced triggering of an allergic response in vitro.The use of recombinant allergens is a promising approach in allergen-specific immunotherapy (AIT). Considerable limitation, however, has been the ability of recombinant allergens to activate effector cells leading to allergic reactions. Recombinant hypoallergens with preserved protein folding and capacity to induce protective IgG antibodies binding effectively to the native allergen upon sensitization would be beneficial for safer AIT. In this study, hypoallergen variants of the major horse allergen Equ c 1 were designed by introducing one point mutation on the putative IgE epitope region and two mutations on the monomer-monomer interface of Equ c 1 dimer. The recombinant Equ c 1 wild type and the variants were produced and purified to homogeneity, characterized by size-exclusion ultra-high performance liquid chromatography and ultra-high resolution mass spectrometry. The IgE-binding profiles were analyzed by a competitive immunoassay and the biological activity by a histamine release assay using sera from horse allergic individuals. Two Equ c 1 variants, Triple 2 (V47K + V110E + F112K) and Triple 3 (E21Y + V110E + F112K) showed lower allergen-specific IgE-binding capacity and decreased capability to release histamine from basophils  It has been argued over a decade that these extracts have shortages mainly because of their inconsistent characteristics and quality. Due to the inherent heterogeneous property of natural allergen sources and extract producing methods, the extracts contain individual allergens with variable content and concentration but also with undefined biological activity. In addition, extracts contain large variety of non-allergenic materials and contaminants which nature is usually unknown and which may have, for example, toxic or undesired immunomodulatory effects. Recombinant allergens produced under good manufacturing practices (GMP) and adequately characterized to show high purity and consistent quality offer several advantages over extracts as medicinal products, most importantly due to the accurate dose administration during AIT4. Protective IgG antibodies capture invading allergen molecules before they reach the effector-cell bound IgE antibodies thus preventing activation of mast cells or basophils2.AIT causes various immunological changes especially in T cells leading to an early increase in allergen-specific IgE response, which decline later and an increase in the allergen-specific protective IgG response, especially subtype IgG1. Peptides or their derivatives have been favored due to their minimal ability to activate effector cells that offers substantial safety factor. However, peptides are prone to rapid degradation by tissue and serum proteases and the use of peptide antigens in immunization experiments is consistently less effective compared to full length proteins3. The use of peptides in immunization generates antibodies which usually recognize denatured (unfolded) proteins whereas immunization with correctly folded proteins produce antibodies which bind to natively folded biologically active proteins3. One approach is to use a peptide carrier strategy which has been reported to induce allergen-specific antibodies that recognize also the peptides in the context of the complete allergens4.A number of clinical trials with recombinant allergens, their variants or allergen-derived peptides with or without carrier proteins have been conducted5. Due to rather challenging techniques associated with isolation of allergen specific IgE antibodies and co-crystallization of protein complexes, only two such IgE/Fab-allergen complex structures have been determined so far; for bovine milk allergen Bos d 5 and for timothy grass pollen allergen Phl p 2. Interestingly, in both of the studies the overall spatial shape of the IgE epitope was flat consisting mainly of secondary structure elements (β-strands) differing from IgG epitopes which are typically located in the protruding regions of allergens7.IgE epitopes are conformational consisting usually several non-continuous stretches (segments) of the polypeptide chain forming a specific three-dimensional structure on the surface of the allergen. The most accurate method to determine an IgE epitope is to co-crystallize the allergen in a complex with the Fab-fragment of patient-derived IgE-antibody6. In our subsequent studies involving a number of allergens with their crystal structures and mass spectrometric analyses, we have discovered that majority of the allergens are able to form weak or transient dimers depending on the allergen concentration8. We have proposed a sequential model for mast cell or basophil activation in which monomeric allergen binds first to the FcεRI bound IgE on the cell surface and subsequent dimerization is strongly boosted due to dimensional reduction  on the cell surface leading to a signal transduction9.The immunocomplex structure of Bos d 5 revealed also that the allergen formed a dimer to which two identical Fab fragments were simultaneously bound indicating that two monoclonal IgE antibodies would be able to form a productive complex with a dimeric allergen to active mast cell or basophil10. Horse allergy is induced by exposure to the horse allergens, such as Equ c 1, c2, c3, c4 and c 5. Of these Equ c 1, the major horse allergen, is a hair dander protein and the most important from a clinical perspective. Equ c 1 is responsible for about 80% of anti-horse IgE antibody response in patients who have been exposed to horse allergens11. Immonen et al. have also analyzed T-cell epitopes of Equ c 1 and found two major, partially overlapping epitopes covering the amino acid residues 140–163  suggesting that dimeric form is predominant14.Equ c 1 belongs to the lipocalin protein family, which members typically transport small hydrophobic ligandsE. coli, purified to homogeneity and validated by size-exclusion ultra-high-performance liquid chromatography (SE-UHPLC) and ultra-high resolution (FTICR) mass spectrometry (MS). IgE-binding profiles of allergens were analyzed by a competitive immunoassay and biological activity by a histamine release assay (HRA).In this study, hypoallergenic variants of Equ c 1 were designed by targeting mutations to the (I) putative IgE epitope region aiming to reduce the binding of IgE antibodies and to the (II) monomer-monomer interface aiming to reduce dimerization of Equ c 1 monomers bound to the IgE-FcɛRI receptor complex yet aiming to preserve the three-dimensional fold highly resembling that of the wild type allergen. Two variants (Triple 2 and 3) of Equ c 1 having one IgE-epitope mutation and two mutations on the monomer-monomer interface allergen were designed. The recombinant Equ c 1 wild type (wt) and variants were produced in 2) was studied by testing different mutations in the middle of the interface in silico. The effect of mutations were predicted by evaluating their ability to break molecular surface complementarity and hydrophobic interactions as well as hydrogen bonds and charged interactions between protein molecules. Hydrophobic groups do not interact well with polar groups, especially with charged ones. Therefore, substitution of hydrophobic residues with charged residues or vice versa should have more substantial effect to diminish protein-protein interaction. It was concluded that V110E and F112K mutations would be the most effective to disrupt the dimer formation of Equ c 1. The IgE epitopes of Equ c 1 are undefined, hence the putative IgE epitope was deduced by searching flat areas on the molecular surface of either side of the two-fold axis of symmetry of the Equ c 1 dimer by using PyMOL. Similarly, it was predicted that mutations E21Y and V47K would be the most promising candidates to achieve the decreased binding of IgE antibodies to Equ c 1. Two variants were selected for further studies: Triple 2 (V47K + V110E + F112K) and Triple 3 (E21Y + V110E + F112K) . The crystal structure contained one molecule in an asymmetric unit. The other half of the Equ c 1 dimer was formed by using the crystallographic symmetry operator. The extensive monomer-monomer interface (area 1024 Å2K) Fig. . Triple SE-UHPLC results show that rEqu c 1 wt, Triple 2 and 3 variants eluted with retention times of 4.0, 4.4 and 4.4 minutes, respectively Fig. . CompareThe high-resolution mass spectrometric characterization of wild-type allergen and the variants Triple 2 and Triple 3 in denaturing solution conditions was used to determine the accurate molecular masses and to observe possible modifications in the protein preparations Figs. . The expNative mass spectrometry was used to study the oligomeric state of proteins and to investigate the correct folding of the hypoallergenic variants compared with rEqu c 1 wt. The native MS showed that at the protein concentration of 40 μM, the rEqu c 1 wt allergen predominantly exists as a dimeric form, whereas the rEqu c 1 variants are mainly monomeric. The native ESI FT-ICR mass spectra displayed a limited number of low charge states, indicating that both the wild-type allergen and the variants have similar, tightly folded structures Fig. .Kd for rEqu c 1 wt dimer was 15.6 ± 0.8 nM which indicates strong tendency for Equ c 1 to dimerize but showing also a nature of this allergen to dissociate to monomers at nanomolar concentrations. It was not possible to perform similar measurements at different concentrations for Triple 2 and 3 because the signal for dimer was very low. However, by using intensities of monomer and dimer of Triple 3 at a single concentration  by using ESI MS. The fitted curve of the free monomeric allergen concentration against the total allergen concentration is shown in Fig. ion Fig. , it can IC50) by fitting a curve to the dilution points of the dilution series of the competing allergen. The trend in the resulting curves were similar irrespective of the spotted concentration used. The results presented here are derived with the spotted wild type allergen concentration of 95 µg/ml.The serum IgE binding to the recombinant Equ c 1 wild type and Triple variants was analyzed by a miniaturized competitive immunoassay Fig. . The wilIC50 values for rEqu c 1 wild type were similar for all sera with the median value of 35 pM  for rEqu c 1 wild type in a competitive immunoassay analysis. These values reflect strong binding of Equ c 1 to specific IgE antibodies of serum and is in agreement with previous studies which showed that IgE antibodies exhibit stronger binding to allergens (KD 10 to 100 pM) compared to IgG binding to antigens (KD 10 to 100 nM)6. Both Triple 2 and Triple 3 variants showed consistently higher IC50 values compared to wild type, median values being 59 and 191 pM, respectively, except for the serum E which showed similar IC50 values between Triple 2 and wt. Reduced binding of allergen-specific IgEs to Triple variants 2 and 3 compared to wt allergen suggests that the mutations V47K (in Triple 2) and E21Y (in Triple 3) are located in the IgE epitope area of rEqu c 1 allergen. E21 seems to be more critical for IgE binding because its mutation produced consistently lower binding compared to V47 mutation. Both residues are located on the flat surface of the antiparallel β-sheet of Equ c 1  for recognition and binding by the IgE antibody-FcεRI complex on the effector cell surface18. It has been earlier shown by Lascombe et al.14 that IgE binding of a serum pool of eight horse allergic individuals to Equ c 1 allergen was efficiently inhibited with only one anti-Equ c 1 monoclonal antibody mAb 220 indicating also that number of IgE epitopes of Equ c 1 is restricted.This kind of flat or planar shape of an IgE epitope has previously been determined for Bos d 5 and Phl p 2 allergens from the immunocomplex structures between the Fab fragment of an IgE antibody and allergenet al.19 who used ultracentrifugation and gel-filtration chromatography and found the strong tendency for Equ c 1 to form dimer but also reported the dimers can be dissociated to monomers by decreasing the protein concentration or lowering the pH of the buffer solution. The strategy to introduce two alterations on the hydrophobic monomer-monomer interface by replacing two hydrophobic amino acid residues by charged amino acid residues proved to be successful since the dimer/monomer ratio of Equ c 1 was dramatically decreased in Triple 2 and Triple 3 as shown by SE-UHPLC and native mass spectrometric analyses. The very narrow charge state distribution in the native mass spectra shows that Triple 2 and Triple 3 are still very compactly folded. The used monomer mutations would thus be effective in the reduction of cross-linking of specific IgE-allergen complexes on the surface of basophils or mast cells.One of the benefits of using native MS for studying oligomers is the detection of all oligomeric forms at the same time which is especially beneficial if protein oligomerization is transient and concentration dependent. Size-exclusion chromatography allows the separation of molecules in aqueous non-denaturing conditions based on their molecular size and shape, therefore enabling the separation of protein monomers from their dimers or aggregates without disrupting protein interactions. The native mass spectrometry and SE-UHPLC analyses indicate that rEqu c 1 wt exists predominantly in dimeric form in micromolar and higher concentrations but is able to dissociate to monomers at lower concentrations. These results are in agreement with the results of Gregoire EC50 values calculated from HRA results were extremely low, 5 to 45 pM (median 30 pM) for rEqu c 1 wt indicating that very tiny amount of this major horse allergen can trigger allergic reaction.EC50 values has been previously reported in some degranulation studies. Blanc et al.20 have reported picomolar median values for peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6, namely 150, 2, 65 and 3 pM, respectively. On the other hand, major birch pollen allergen Bet v 1 seems to trigger degranulation at the higher concentrations, approximately in 2 nM21. This may indicate differences in the capacity of natural allergens to cross-link IgEs on the surface of basophils.EC50 values  obtained for rEqu c 1 wt is in agreement with low IC50 values obtained from the competitive inhibition study , which explains that the potency of rEqu c 1 wt to trigger degranulation at extremely low concentrations is largely based on a strong binding of IgE antibodies to allergen. On the other hand, the increase in IC50 values was smaller for Triple 2 (1.7 fold increase) and Triple 3 (5.5 fold increase) compared to EC50 values for Triple 2 (7.7 fold increase) and Triple 3 (15.7 fold increase). Because IC50 values reflects the binding of IgE antibodies, the higher increase in EC50 values can be explained by two mutations (V110E + F112K) on the monomer-monomer interface of Equ c 1. Altogether, three point mutations, one on the putative IgE epitope and two on the monomer-monomer interface of Equ c 1 have thus successfully reduced the formation of dimeric allergen-IgE complexes and subsequent cross-linking of IgE on the cell membrane of basophils.The low et al.1, recombinant allergen molecules have several advantages over natural extracts in AIT. In addition, it has been demonstrated that full length folded proteins are more effective in developing antibodies that recognize native structures3. It can be thus argued that natively folded recombinant allergen molecules or their derivatives with preserved native structure would be promising candidates in AIT. Phase II studies have been reached with recombinant wild type allergens from birch (Bet v 1) and timothy 1. The results have been positive but not so much superior that these products would have been further developed into market.As reviewed recently by Valenta The key issues in the development of novel recombinant allergens or their derivatives for AIT are their efficacy and safety. One pathway forward to create a safe and efficient hypoallergen is to mutate a limited number of amino acids in allergen affecting its allergenicity yet preserving the genuine folded three-dimensional structure. Thus, the release of allergy mediators from mast cells and basophils and the consecutive appearance of allergic symptoms would be reduced. Instead, protective IgG antibodies against the wild type allergen would be induced. This would prevent mast cell and basophil activation and lead to the modulation of immunoresponse. However, this requires information on critical amino acid (hot spot) residues which especially contribute to the productive formation of allergen-IgE complex on the surface of mast cell or basophil.12. Amino acid sequence identity between Equ c 1 Triple 3 and wild type is extremely high, 98.4%, which considerably increases the possibility that IgG antibodies induced by Triple 3 would efficiently bind to Equ c 1 wt. Additionally as shown here, the Triple 3 was recombinantly produced and purified resulting in high purity, homogeneity and correct fold to support the steps toward consistent batch-to-batch manufacturing and safety of Triple 3 as medicinal product. Altogether this would make Triple 3 variant promising candidate for AIT in horse allergy. To evaluate the suitability of Triple 3 further for clinical studies, it would be important to test IgE-binding and histamine release with a wider panel of serum samples of individuals allergic to Equ c 1, to perform T-cell reactivity tests, and continue to animal studies.Equ c 1 Triple 3 variant that contains three point mutations, one on the putative IgE epitope and two on the monomer-monomer interface, of the major horse allergen Equ c 1 has resulted in reduction of histamine release when using sera from five horse allergic patients. The mutations are not located in the region which was previously reported to include two major T-cell epitopes22.Three-dimensional structure of protein molecules were analyzed and visualized using open-source PyMOL 1.7 software. Molecular surfaces were created using UCSF ChimeraX softwareEscherichia coli BL21(DE3) (Novagen) strain.Synthetic gene fragments encoding Equ c 1.0101 (thereafter wild type or wt) and Equ c 1 variants V47K + V110E + F112K (Triple 2) and E21Y + V110E + F112K (Triple 3) with codon optimization for bacterial expression were purchased from GenScript. The synthetic gene fragments of Equ c 1 wt and Triple variants were cloned as NcoI-NotI restriction fragments under the T7 promoter of the pET-28b(+) vector (Novagen) for cytoplasmic expression. The recombinant Equ c 1 allergens contain an extra alanine residue in the N-terminus enabling the usage of NcoI restriction site as well as to keep the open reading frame intact in the cloning of the synthetic genes. The expression vectors were transformed into the 600 reached ~1. Protein expression was induced by addition of IPTG  to a final concentration of 1 mM, and cultivation was continued for 4 h at +37 °C with 220 rpm shaking. Cells were harvested by centrifugation . The insoluble protein fraction was isolated from the cell pellet according to Hoffmann-Sommergruber et al.23 and refolding of the allergen polypeptides according to Arango et al.24 and Stancombe et al.25 Briefly, collected bacterial cells were resuspended in 25 mM imidazole, 0.1% TritonX-100 pH 7.4 and lysed by three cycles of freezing and thawing steps. Inclusion bodies were isolated by centrifugation and solubilized in 6 M Urea, 10 mM CAPS pH 10.5 followed by a 3-hour incubation at room temperature under slow stirring. Urea was removed by slowly diluting the solubilization solution 1:10 (v/v) in 10 mM CAPS pH 10.5, after which pH was adjusted to 7.5. Protein was allowed to refold for minimum of 66 hours at +4 °C before concentration with Prep/Scale-TFF 6 ft2 10000 MWCO Cartridge (Millipore).Recombinant Equ c 1 wt and Triple variants were produced in 1.8 L (6 × 300 ml) shake flask cultivations in LB medium supplemented with kanamycin (25 µg/ml) and cultivated at +37 °C with 220 rpm shaking until OD280 and using sequence-derived extinction coefficient.Recombinant Equ c 1 wt and Triple variants were purified by a standard ion exchange  followed by a size-exclusion chromatography . Elution peak fractions after final purification step were analyzed by a Coomassie-stained SDS-PAGE showing that obtained proteins were of high purity and homogeneity. Concentrations of the pure allergens were determined by measuring A2HPO4, 3 mM NaH2PO4, 150 mM NaCl, pH 7.3) as a mobile phase at a flow rate 0.3 ml/min until the baseline was stable. Samples of 2 µl of a 40 µM protein solution were injected. The chromatographic separation was carried out under isocratic flow with overall run time of 12 minutes and detection at 214 nm wavelength. The BEH125 SEC Protein Standard Mix (Waters) was used as a gel filtration standard.Size-exclusion ultra-high-performance liquid chromatography (SE-UHPLC) analysis of rEqu c 1 wt and Triple variants were performed using Acquity BEH125 SEC column with dimensions of 4.6 × 150 mm, a pore size of 125 Å and a particle size of 1.7 µm (Waters) coupled with Acquity I-Class UPLC instrument (Waters) and controlled by Empower 3 software (Waters). The column was equilibrated to running conditions with PBS (12 mM Na−1. The temperature and flow rate of the drying gas (N2) were 200 °C and 4.0 L/min, respectively, and the pressure of the nebulizer gas was 1.0 bar. Mass spectra were acquired in the positive-ion mode over the m/z range of 800–3000 with 1-Mword time-domain transients and 0.05 s ion accumulation time.The mass spectrometric analyses were carried out with a 12 T SolariX XR FT-ICR mass spectrometer, equipped with an Apollo II ion electrospray ionization (ESI) source . Protein samples were desalted and exchanged into an ESI-compatible ammonium acetate buffer  using PD-10 desalting columns . For mass spectrometric characterization in denaturing solution conditions, samples were diluted to the concentration of 0.2 μM with acetonitrile/water/acetic acid solution . The protein samples were directly infused into the ion source by a syringe pump at a flow rate of 2 μl minFor studying proteins in their native state, the instrumental parameters were carefully optimized to maintain the weak, non-covalent interactions in the gas-phase. Proteins were measured in 10 mM ammonium acetate buffer (pH 6.9). The drying gas temperature was set to 80 °C, and ion accumulation time was 1.0 s. For a native mass spectrum, 100 co-added 262-kword time domain transient were collected and processed to 524-kword data. The wild-type allergen and its Triple variants were measured by using the same instrumental parameters, in order to avoid any bias between the samples. Mass spectra were calibrated externally with respect to the ions of ES Tuning Mix . Data collection was controlled by ftmsControl software and the data were processed by using Data Analysis software .Kd) of the rEqu c 1 wild type allergen, a dilution series with different protein concentrations was prepared. The concentrated stock solution of rEqu c 1 allergen was diluted with 10 mM ammonium acetate to the (monomer) concentrations of 0.1 to 5.0 μM. The slow dissociation of dimers to monomers was taken in consideration by letting the diluted samples to stay four days at +4 °C. The actual concentration of the diluted samples was calculated just before the mass spectrometric analysis by the measured absorbance at 280 nm, using the sequence-derived extinction coefficient. The most diluted samples with concentrations of 0.1 and 0.25 μM showed very low absorbances, suggesting that the protein molecules were partially adsorbed to the walls of the plastic eppendorf tubes. For the less diluted protein samples, five parallel native mass spectra were measured at each different protein concentrations.For a determination of the dissociation constant  was determined from the absolute intensities of ions representing either the monomer (P) or the dimer (P2). The total intensities for P and P2 were calculated by combining the peak intensities over the charge state distribution (z = 1 to n) in the ESI-MS spectra. The intensity ratio sufficiently reflects the concentration ratio in solution, if the ionization and transmissions efficiencies are similar for the monomer and dimer. Kd for rEqu c 1 wt dimer was obtained using the procedure described in detail by Niemi et al.9.The utilization of native ESI-MS for determining monomer-dimer ratio of (transient) protein complexes has been discussed in literatureFor the competitive IgE binding assay rEqu c 1 wild type was biotinylated according to the manufacturer’s instructions using a 10-fold molar excess of EZ-Link Sulfo-NHS-LC-Biotin reagent (ThermoFisher Scientific). The biotinylated Equ c 1 wt was spotted as three concentrations  onto the streptavidin coated microtiter plate wells (ThermoFisher Scientific) by Nano-Plotter 2.1 (GeSiM). Serum samples from five horse allergic individuals were selected based on their ImmunoCAP ISAC (ThermoFisher Scientific) test profiles. The serum samples used in the competitive immunoassay and histamine release assay were collected from volunteer horse allergic adult (over 18 years) individuals after the permission of the Ethical Committee of Helsinki University Hospital (Finland). The optimal dilution for each serum chosen to be used in the competitive assay was analyzed by IgE serum titration analysis for each spotted concentration (data not shown). For competitive assay, diluted serum was pre-incubated with different amounts of competing allergen in final allergen concentrations of 10 000, 2 000, 400, 80, 16, 3, 0.6 and 0 pM and then added to the well in which the biotinylated rEqu c 1 wt was immobilized. All of the experiments were performed in duplicates.Peroxidase-conjugated anti-human IgE (Southern Biotech) was used for secondary detection and fluorescently-labelled tyramide (ThermoFisher Scientific) for signal amplification. The fluorescence readout was obtained by scanning the wells with LS400 microarray scanner and 633 nm laser (Tecan). Image analysis, spot detection and fluorescence intensity quantification were performed with Array-Pro Analyzer software (Media Cybernetics). Local background signal value was measured outside the specific spots from each well. The net signal value for each spot was obtained by subtracting the local background signal value from the average intensity value of each spot.27.Biological activity of the rEqu c 1 wild type and Triple variants was analyzed by a histamine release assay (HRA). HRA was performed as an outsourced service at RefLab Aps . Briefly, stripped human basophils were passively sensitized with rEqu c 1 wt and Triple variants in 16 different concentrations from 0.05 fM to 0.5 nM (1fg/ml to 10 ng/ml) using the same five serum samples (A-E) as in the competitive IgE binding assay and with an additional positive control serum sample (F) provided by RefLab. Released histamine induced by different allergen concentrations was measured as duplicates by ELISA (enzyme-linked immunosorbent assay) using a glass microfibre method developed by RefLab. In this method glass microfibres coated on microtiter wells bind histamine released from allergen triggered basophils with high affinity and selectivityP value of less than 0.05 was considered statistically significant.Graphical display, curve fitting, Student’s t-test analysis of competitive ELISA  and histamine release assay data were done with GraphPad Prism v. 8.2.1 (GraphPad Software Inc.). A Human participants are not directly involved in this study. The origin of the human serum samples used in the competitive immunoassay and histamine release assay (HRA) and the human basophils utilized in HRA is described as follows. The serum samples used in the competitive immunoassay and histamine release assay were collected from volunteer horse allergic individuals after the permission of the Ethical Committee of Helsinki University Hospital (Finland). Serum samples were collected by Desentum Ltd as an outsourced service using healthcare professionals. The blood samples were numbered and the identity of the donors is known only by the responsible scientist and the healthcare professionals who participated in the collection of the blood samples. Biological activity of the recombinant allergens was analysed by the histamine release assay as an outsourced service at RefLab ApS . RefLab is an accredited laboratory according to: EN ISO/IEC 17025: 2005 and has an in house assay for measuring histamine release from basophils obtained from blood bank buffy coats. Buffy coats were collected at the Blood Bank, National University Hospital of Copenhagen. The Blood Bank has a general ethical approval to hand out buffy coats making sure that the blood donors are anonymous. It is therefore not possible to obtain case history or laboratory case records of the blood donors. RefLab has a contract with the Blood Bank allowing using the buffy coats. The study was conducted in compliance with the Declaration of Helsinki.Supplementary Information.


A range of flavan-3-ols, ellagitannins and chemically defined proanthocyanidin fractions was evaluated in comparison to oseltamivir carboxylate and zanamivir for their inhibitory activities against viral influenza A (H1N1) and bacterial Vibrio cholerae neuraminidase (VCNA). Compared to the positive controls, all tested polyphenols displayed a weak inhibition of the viral enzyme but similar or even higher potency on the bacterial neuraminidase. Structure–activity relationship analyses revealed the presence of galloyl groups and the hydroxylation pattern of the flavan skeleton to be crucial for inhibitory activity. The combination of zanamivir and EPs® 7630 (root extract of Pelargonium sidoides) showed synergistic inhibitory effects on the bacterial neuraminidase. Co-crystal structures of VCNA with oseltamivir carboxylate and zanamivir provided insight into bacterial versus viral enzyme-inhibitor interactions. The current data clearly indicate that inhibitor potency strongly depends on the biological origin of the enzyme and that results are not readily transferable. The therapeutic relevance of our findings is briefly discussed.Neuraminidase is a key enzyme in the life cycle of influenza viruses and is present in some bacterial pathogens. We here assess the inhibitory potency of plant tannins versus clinically used inhibitors on both a viral and a bacterial model neuraminidase by applying the 2′-(4-methylumbelliferyl)-α- Influenza is an acute viral infection of the respiratory tract, afflicting millions of individuals each year. Due to the imminent threat of epidemics and pandemics in human population and the alarming emergence of drug-resistant influenza virus strains, there is an urgent need for new and effective anti-influenza drugs. The influenza virus employs two key enzymes located on the surface, hemagglutinin (HA) and neuraminidase (NA), to initiate viral fusion and subsequent budding of progeny virions from the infected cell . In the Annual vaccination has been the main global strategy for preventing influenza infections, but frequent virus antigenic drifts present a permanent challenge in the development of effective vaccines. Antiviral drugs offer an alternative therapeutic option. Currently, there are two classes of anti-influenza drugs available, matrix-2 (M2) protein ion channel blockers and neuraminidase inhibitors (NAIs). The therapeutic efficacy of M2 ion channel blockers such as amantadine and rimantadine is limited to influenza A viruses. These therapeutics are also associated with the rapid emergence of drug resistance and have the disadvantage of side effects. At present, the approved NAIs oseltamivir, zanamivir, peramivir, and laninamivir are first choice antiviral drugs for the treatment of influenza A and B virus infections. Until the season 2007/2008, NAI-resistance to the first introduced NA-targeting drugs has been reported only sporadically [A successful modus operandi for finding promising antiviral agents involves the exploration of plants and their chemical constituents. The plant kingdom has been recognized as an inestimably rich source of metabolites for target-oriented drug discovery. In particular, various plant polyphenols that have a broad spectrum of other biological activities have been identified to possess viral NA inhibitory activities ,4,5. AltStreptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae [NAs are also present in other biological systems , for exacholerae ,11,12. Bcholerae . Their fcholerae . Taking cholerae , bacteriEstablished functional assays to assess NA inhibitory activities are based on either fluorescence or chemiluminescence and are reported to be compared to culture-based assays, more predictive in terms of their in vivo susceptibility . The in To fill this gap, we here disclose results for the inhibition of both a viral and a bacterial model NA by various tannins. In addition to a range of flavan-3-ols and ellagitannins, highly purified and chemically defined proanthocyanidin fractions of different composition were included to gain insight into structure–activity relationships for this group of polyphenols. To provide a rationale for differential inhibition of bacterial and viral NAs, we additionally performed X-ray crystallographic analyses of NAs in complex with the synthetic reference compounds oseltamivir carboxylate and zanamivir.d-N-acetylneuraminic acid (MUNANA)-based activity assay [Preliminary experiments in our research group have indicated differential inhibition of bacterial and viral NAs by some polyphenolic substances  and havety assay . The assVibrio cholerae neuraminidase (VCNA). With IC50 values (the inhibitor concentration that is required for 50% inhibition) of around 10 nM, oseltamivir carboxylate and zanamivir are very active against the viral H1N1-NA. The bacterial VCNA, on the other hand, is only moderately inhibited by the reference compounds, displaying IC50 values of 144 µM for oseltamivir carboxylate and 52 µM for zanamivir. These IC50 values are comparable to other NA inhibition data [To validate our assay conditions, we first assessed the inhibitory potency of the reference NAIs oseltamivir carboxylate and zanamivir  against ion data ,20, alth50 values range from 0.3 to 0.9 mM, and gallocatechin-3-O-gallate is the most active compound within this series. This finding is consistent with modest inhibitory activities (>100 µM) reported for (epi)catechin [The inhibitory potencies of a series of flavan-3-ols are shown in O-gallate (IC50 = 25 µM), catechin-3-O-gallate (IC50 = 55 µM), and epigallocatechin-3-O-gallate (IC50 = 64 µM) have comparable or greater potency compared to the reference compounds  and terchebin (IC50 = 31 µM). In tests with the viral H1N1-NA, the inhibition is also in the µM range, but terchebin showed a 3.5-fold higher inhibitory potency compared to paeonianin C , corresponding to three, one, and no galloyl groups. Further examination of the structures revealed that the presence of additional 1′,6′- or 3′,6′-hexahydroxydiphenoyl (HHDP) residue  unit in addition to a varying number of galloyl groups on the glucose core. Structure–activity relationship analyses indicate that the inhibitory activity of the tested compounds depends in part on the degree of galloylation. As shown in  residue  results C-glycosidic ellagitannins was limited, some structural features determining inhibitory potency were evident. Paeonianin C, possessing a 1C-penta-O-galloyl glucose moiety, was significantly more potent (IC50 = 15 µM) than vescalagin (IC50 = 73 µM), having a 4,6-HHDP and a 2,3,5-flavogalloyl unit, and casuariin (IC50 = 236 µM), which lacks the 5-O-galloyl residue as part of the flavogalloyl group. The inhibition correlates with the number of pyrogallol elements. More information is needed to decide between galloyl-derived structural variants. However, it appears that the conformation of these molecules with bulky substituents plays a significant role and more C-glycosidic ellagitannins need to be tested to fully understand the structure–activity relationships.Although the number of tested 50 values for both viral H1N1-NA and bacterial VCNA inhibition. Most of the tannin fractions inhibit the bacterial VCNA significantly better than the reference compounds. The inhibition of viral H1N1 is reduced by a factor of 6–10 compared to the clinically used inhibitors, leaving potential for further optimization.Proanthocyanidins are oligomeric flavan-3-ols and represent another group of plant tannins. Extensive structural variations and challenges in the isolation and purification of chemically defined proanthocyanidins prompted the evaluation of well-characterized plant fractions. Diospyros kaki sample is the most active fraction, suggesting that pyrogallol B-ring elements (prodelphinidin units) and 3-O-galloylation are preferred structural determinants for NA inhibition. In line with this, weaker inhibitory activity is found for the prodelphinidin-rich but mainly ungalloylated extract of Pelargonium sidoides EPs® 7630 extract. The trend continues for the Nelia meyeri and Salix spp. fractions, which comprise either homogeneous 2,3-cis or 2,3-trans proanthocyanidins composed of non-galloylated dimers to hexamers in similar proportions. Interestingly, fractions with mixed 2,3-trans and 2,3-cis constituent flavanyl units obtained from Potentilla erecta and Betula spp. are less potent than preparations dominated by compounds with the homogeneously linked building blocks. Increasing proportions of 2,3-cis entities as in the Betula fraction apparently reduces the inhibitory activity  , the slope of the concentration–effect relationship , and the linear regression correlation coefficient  were determined from the median–effect plots. The parameter r of the median–effect plots exceeds 0.95 (data not shown), showing the conformity of the data to the median–effect principle. The calculated mD values indicate an enhanced VCNA inhibition with increasing amounts of EPs® 7630  as compared to zanamivir alone (mD = 17). The enzyme was treated with zanamivir and EPsor 10:1) . The med50, IC75, IC90, and IC95, indicating the concentrations required to achieve 50%, 75%, 90% or 95% inhibition of VCNA activity. It should be noted that only CIs of higher effect levels  are considered relevant for therapeutic applications [af-CI plots for various zanamivir/EPs® 7630 combinations are shown in af values in our study. For higher effect levels, CI values move to synergism except for the 1:1 combination, exhibiting continuously antagonistic effects (wt) was calculated on the basis of selected average CI values including IC50, IC75, IC90 and IC95 at four representative effect levels (af > 0.5). The most effective combinations include drug–herb ratios of 10:1, 5:1, and, surprisingly, 1:10, with CIs ranging between 0.5 and 0.8, indicative of synergism. For the zanamivir/EPs® 7630 combination with a ratio of 1:5, the CI revealed additivity. We calculated the combination index (CI) values that offer a quantitative definition for an additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in drug combinations ,28. CI vications . The fa- effects . The com® 7630  and zanamivir  alone and for the tested combinations  indicate possible different binding sites for each compound. Therefore, the data were reanalyzed with an assumption of non-exclusivity for the calculations of CI values of the tested combinations (wt) are very similar for all the tested combinations, whether the calculation was based on exclusivity or non-exclusivity. Further analyses of the complex EPs® 7630 extract composition [Differences in the slopes of the median effect plots for EPsinations . Howeverposition ,31 mightThe in vitro studies presented here unambiguously show the different inhibitory potency of tannins against bacterial and viral NAs. To better understand the differential NA–tannin interactions at the molecular level and to assess the specificity of tannin binding, we aimed at solving the crystal structures of various NA–flavan-3-ol complexes. These attempts, however, remained unsuccessful. Instead, we solved the crystal structures of bacterial VCNA in complex with the reference compounds oseltamivir carboxylate and zanamivir at 1.87 and 1.75 Å resolution , allowinThe uncomplexed crystal structures of influenza A H1N1-NA and bacterial VCNA have been previously published. The enzymes have different overall structural topologies. The viral enzyme is a mushroom-like shaped homotetramer, while the bacterial NA is a monomer with two flanking lectin-like domains ,33,34. BA common feature of viral and bacterial inhibitor binding is the electrostatic and hydrogen bonding interaction of the carboxylic acid group of oseltamivir carboxylate or zanamivir to a cluster of three positionally conserved arginine residues  . Notably50 of 52 µM versus 144 µM, H-pyran ring of zanamivir forms a hydrogen bond with the side chain of Tyr740, while the cyclohex-1-ene entity of oseltamivir carboxylate interacts exclusively hydrophobically  [50 of 52 µM versus 144 µM, The impact of oseltamivir carboxylate and zanamivir on the activity of other bacterial NAs has been reported in previous publications ,42,43. Tanamivir . Also th® 7630 is a more potent inhibitor of bacterial VCNA than either compound alone, suggesting promising benefits of similar combinatory therapies. Clinical studies are needed to approve the therapeutic potential. The fact that herbal medicines are increasingly used as an adjuvant treatment of both bacterial and viral infections makes this field a promising but challenging research in the future.Another important finding of our study is that the drug–herb combination zanamivir and EPsFinally, absorption, distribution in tissues, and metabolism are important efficacy-influencing parameters. The intestine and its microbiota play a crucial role in this process. Further research is needed to clarify the impact of microbial degradation of tannins and their metabolites on health effects.d-N-acetylneuraminic acid (MUNANA), and 2-(N-morpholino) ethanesulfonic acid (MES) were purchased from Sigma Aldrich (Taufkirchen Germany). Sources of NAIs: Oseltamivir carboxylate (5-N-acetyl-3-(1-ethylpropyl-1-cyclohexene-1-carboxylic acid), the active form of oseltamivir, was kindly supplied by Hoffmann La Roche  and zanamivir  by GlaxoSmithKline . All flavan-3-ols (purity > 95%) were commercially obtained , while the ellagitannins are generous gifts from Prof. T. Yoshida, Okayama University, Japan. The highly purified proanthocyanidin fractions obtained from Nelia meyeri, Salix spp., Betula spp., Potentilla erecta, Rhus leptodictya, and Diospyros kaki were available in our research group. The preparation and characterization of the oligomeric proanthocyanidin mixtures are described elsewhere [Pelargonium sidoides , was obtained from Dr. Willmar Schwabe Pharmaceuticals .4-Methylumbelliferone, 2′-(4-methylumbelliferyl)-α-lsewhere ,48,49,50Vibrio cholerae NA was performed [2) and stored at −22 °C in aliquots, while the viral NA  in Tris-buffer  was adjusted to 80–120 mU/mL with distilled water and kept in aliquots at −80 °C until use. Stock solutions (0.1–10 mg/mL) of the test substances were prepared in MES-buffer, aqueous ethanol or DMSO/H2O (1:5 v/v) and serially diluted in MES-buffer for measurements.To assess NA activity, a fluorometric NA inhibition assay using MUNANA as a substrate and commercially available erformed . VCNA (Hn = 3–6) and IC50 values for each substance were calculated from fitting curves to data points (percentage of inhibition vs. concentration) using GraphPad Prism version 6.0 for Windows . To quantify enzyme activity, 60 µL of MES-buffer and 10 µL of diluted VCNA were mixed with 10 µL of varying concentrations of the test substance in black 96-well plates. For analyses of the viral NA, half of the volumes were used. After agitating gently and incubating for 2 min at 37 °C, 20 µL of the substrate MUNANA (1 mM stock solution) was added, and the fluorescence measured over the course of 14 min at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Appropriate controls were included in the measurements. All assays were at least performed in triplicates  was applied, followed by the t-test. For data with non-normal distributions, the Welch-modified t-test was used. Analysis of variance (ANOVA) followed by the Bonferonni’s multiple comparison test was used to evaluate differences between tests. p-values <0.05 were considered statistically significant.IC® 7630 on VCNA was determined using the diagonal constant ratio combination method designed by Chou and Talalay [® 7630 individually and in combination at weight ratios of zanamivir to EPs® 7630  as described in m), intercepts and regression coefficients were used to determine the median effect dose Dm).The combined effect of zanamivir and EPs Talalay . VCNA waaf) and fraction unaffected (uf) by the concentration D were calculated by the median effect equation [Fraction affected  NA was a gift from Prof. G. Taylor, University of St. Andrews, Scotland. VCNA was produced according to a previously reported method with the following modifications [E. coli Rosetta™ 2 (DE3)  using an LEX ultra-high-throughput bench-top bioreactor . Cells were grown at 37 °C in Terrific Broth medium to an optical density at 600 nm (OD600) of about 2.0–2.5, cooled to 17 °C, and induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). Cultures were then left shaking overnight, and cells were collected by centrifugation. The expression vector pET30b(+) that produces ications . VCNA waw/v), desalting on a 26/10 HiPrep Desalting column  equilibrated with 10 mM Tris-HCl pH 7.6 and 150 mM NaCl, a Source 30Q anion exchange column, and a final size-exclusion chromatography on a Superdex 200 prep grade column . Purified protein was concentrated to about 6 mg/mL in a buffer containing 20 mM Tris-HCl, pH 7.6, 0.15 M NaCl, and 10 mM CaCl2.For purification, cells were resuspended in 20 mM Tris–HCl pH 7.6 supplemented with cOmplete™ ethylenediamine tetra-acetic acid (EDTA)-free protease inhibitor cocktail , and lysed by sonication . The purification procedure comprised initial ammonium sulfate precipitation (50% 2) was complexed with a 20 mM inhibitor  and concentrated to 6–7 mg/mL. The VCNA–oseltamivir complex was crystallized using 20% (w/v) polyethylene glycol (PEG) 3350 and 0.2 M lithium acetate as reservoir solution. For the VCNA–zanamivir complex, the reservoir solution contained 20% (w/v) PEG 3350 and 0.2 M sodium fluoride. Before flash-freezing in liquid nitrogen, the crystals were transferred into a cryoprotectant consisting of reservoir solution supplemented with 30% (v/v) glycerol. The VCNA-inhibitor complexes were crystallized using the sitting-drop vapor-diffusion method at 20 °C by mixing equal volumes (200 nL) of purified protein see  and reseXdsapp [Phaser [Phenix program suite [Coot was used for manual model building and visualization [Prodrg was used for generation of ligand restraint parameters [X-ray diffraction data were collected at beamline BL14.1 at the Helmholtz-Zentrum Berlin  at a wavXdsapp . The str [Phaser  and unco [Phaser . Structuam suite . The gralization . Prodrg rameters . Data coVibrio cholerae NA and influenza virus H1N1 A/California/04/2009 NA are P0C6E9 and C3W5S3.The structure-based sequence alignment was performed with PDBeFold  using th