A large retreat of sea-ice in the ‘stormy’ Atlantic Sector of the Arctic Ocean has become evident through a series of record minima for the winter maximum sea-ice extent since 2015. Results from the Norwegian young sea ICE (N-ICE2015) expedition, a five-month-long (Jan-Jun) drifting ice station in first and second year pack-ice north of Svalbard, showcase how sea-ice in this region is frequently affected by passing winter storms. Here we synthesise the interdisciplinary N-ICE2015 dataset, including independent observations of the atmosphere, snow, sea-ice, ocean, and ecosystem. We build upon recent results and illustrate the different mechanisms through which winter storms impact the coupled Arctic sea-ice system. These short-lived and episodic synoptic-scale events transport pulses of heat and moisture into the Arctic, which temporarily reduce radiative cooling and henceforth ice growth. Cumulative snowfall from each sequential storm deepens the snow pack and insulates the sea-ice, further inhibiting ice growth throughout the remaining winter season. Strong winds fracture the ice cover, enhance ocean-ice-atmosphere heat fluxes, and make the ice more susceptible to lateral melt. In conclusion, the legacy of Arctic winter storms for sea-ice and the ice-associated ecosystem in the Atlantic Sector lasts far beyond their short lifespan. Similarly, four of the last five Arctic winters were among the warmest on record12. The most pronounced retreat of Arctic winter sea ice is in the Barents Sea, where sea ice has thinned15 and there has been a 47% reduction in ice extent over the satellite record14. The retreat of winter sea ice in the Barents Sea and north of Svalbard also coincides with the path of the warm Atlantic Water inflow17. This inflowing Atlantic Water has warmed and shoaled in recent years, and is likely a key mechanism of sea ice loss in the region20.Following the rise in winter storm activity25. However, the full impact of winter storms on the Arctic remains unclear. In-situ measurements, of any type, from the central Arctic Ocean are scarce during winter. Therefore, earlier studies have relied heavily on atmospheric reanalyses, numerical models, remote sensing products, weather station data from the peripheries of the Arctic Ocean, or autonomous buoys. Such approaches present a number of problems. For example, reanalyses suffer from large temperature biases in the Arctic during winter, and there is a substantial spread in simulated precipitation products27. Uncertainties in snow depth on sea ice can induce large errors in ice thickness measurements from remote sensing30, and buoys reflect point observations in a system with large spatial variability over small distances32.Many studies have investigated the impact of summer and winter storms on Arctic sea ice33  campaign took place in an area of relatively thin (<1.5 m) pack ice north of Svalbardet al.34 documented how the N-ICE2015 camp observed six major winter storms (M1-M6), from 20 January and 21 March 2015 . The northerly winds advected cold and dry, polar air, over the camp36  thermodynamic growth on the underside of snow-covered first and second year sea ice between January and March 2015, despite prolonged periods with air temperatures below −30 °C42 Fig. . In cont46 . Specifically, we perform two simulations. In the first simulation, we force the model with the total precipitation from an atmospheric reanalysis. This produces a 0.4 m snowpack in January, which is consistent with N-ICE2015 observations39. However, the extreme transient warming events during N-ICE2015 winter storms had limited impact on growth rates of snow covered sea ice  can cause overestimation of sea ice thickness by space-borne radar altimeters such as CryoSat-250. King et al.30 showed that radar reflections from CryoSat-2 in the N-ICE2015 region during spring 2015 were closer to the snow freeboard than ice freeboard, and resulted in a systematic overestimation of sea-ice thickness by a factor of two. This highlights the need to give careful consideration of snow properties when interpreting satellite radar altimetry data in this region.We note that the presence of saline snow and also the internal structure of the snow pack  could instead act to reduce the future snow load on sea ice and decrease the potential for snow-ice formation53.We speculate that continued thinning of Arctic sea ice may increase the potential for future flooding and snow-ice formation in the Atlantic Sector of the Arctic Ocean. Nonetheless, historic and projected trends in Arctic precipitation are highly uncertain54. This compacted the ice pack and increased the ice concentration around the N-ICE2015 camp , during (9%), and after (28%) storm M2  is found close to the surface . We find that each N-ICE2015 winter storm is associated with a reduction in sea-ice concentration of up to 25%, over the Atlantic Water inflow path north of Svalbard Fig. . After i M2 Fig. . During s38 Fig. . Sea-ices38 Fig. , presuma2 concentrations observed at the N-ICE2015 camp during and after storms M2 and M3, signals deeper CO2 and nutrient rich water being mixed upwards57 . It is commonly understood that the spring algae bloom can only develop after the light-attenuating snow layer melts69. However, late spring observations from N-ICE2015, made in the same region but over different ice floes, showed that the increased light transmission through the numerous leads was sufficient for the development of an early under-ice phytoplankton bloom during the pre-melt season72  are likely to have a more negative impact on winter ice growth, compared with storms later in the season. This is due to a combination of the lasting effects of snow insulation, and the fact thinner sea ice is more sensitive to the effects of snow insulation than older thicker ice−2 to be widespread over the Arctic Ocean25. These heat fluxes are unlikely to cause bottom melt, but will reduce winter ice growth. Large ocean heat fluxes capable of causing rapid bottom melt are a regional phenomenon unique to the Atlantic Water inflow path. Similarly, we only expect snow-ice formation to occur in the Atlantic Sector of the Arctic Ocean, where winter precipitation totals are sufficiently high27 relative to the thinning sea ice to make it susceptible to flooding42.While the observations presented in this paper are from storms in the Atlantic Sector, many processes we describe will be applicable for winter storms in other regions of the Arctic Ocean Fig. . Specifi3. While winter storm activity has increased here in recent years, it is unclear whether this trend will continue into the future80. Nevertheless, the overall impact of winter storms on sea ice will likely increase in a warmer Arctic. With a thinner sea-ice cover76 and warmer and shallower Atlantic Water19, the tight air-snow-ice-ocean coupling we observed during storms will strengthen. Therefore, we expect more break-ups and ridging, more leads, greater ocean heat fluxes, faster melt, and possibly more flooding in response to winter storms. These emerging physical properties of the new Arctic sea ice regime will also have consequences for the ice-associated ecosystem.The N-ICE2015 observations from the Atlantic Sector of the Arctic Ocean are especially important because this region experiences the most frequent and strongest winter stormsIn-situ observations are thus essential to improve our understanding of these processes. We emphasize that the autumn-freeze up and winter season are important periods to target in future field campaigns as it sets the scene for the summer melt period.The spatial scales of many processes highlighted above are typically small (<1000 m). This hampers the detection of these processes from remote sensing and ultimately their representation in climate models. et al.33.The Norwegian young sea ice expedition N-ICE2015) took place in the sea ice pack north of Svalbard from January – June 2015, onboard the Research Vessel Lance . Mean sea level pressure was measured with an RM Young 61302 V pressure sensor at the base of the mast. The 2 m air temperature was measured with a Vaisala HMP155 sensor mounted inside a triple-walled actively aspirated radiation shield (RM Young Model 43502). We averaged these processed data from 10-minute resolution to six-hour means . Hence, the precipitation measurements are likely to be biased low due to blowing snow, especially during storm periods. For further details about these datasets, quality control, and uncertainties, refer to Cohen et al.34. For comparison with the precipitation observations, we also present the total daily precipitation from the European Centre for Medium Range Weather Forecasting’s Interim Re-Analysis (ERA-Interim)83 at the ice camp position. The timing of precipitation events in the Arctic is known to be very consistent among reanalyses, although there is a spread in the absolute values of total precipitation27. Note that we use the total precipitation product and not the snowfall. This is because a significant fraction of the total precipitation in the central Arctic is classified as rain, even during midwinter and sub-zero temperatures in ERA-I46. Downward longwave radiative fluxes84 were first presented by Walden et al.37. These were measured using a heated and ventilated Kipp and Zonen CGR4 pyrgeometer37. We averaged the processed data files from 1-minute resolution to six-hour means  were launched from the ice camp daily at 1100 and 2300 UTC36.The N-ICE2015 meteorological observationset al.34. These storms (M1 – M6) were defined locally at the N-ICE2015 camp, as periods with a rapid pressure drop of at least 5 hPa/6 hrs and sustained wind speeds of more than 7 m s−1 , developed by the Scottish Association for Marine Research15f Fig. . The int88. Itkin et al.54 calculated the mean ice drift speed in Fig. 88.The ice drift speed in Fig. 90, for a case study of N-ICE2015, covering the period September 2014 – March 2015. The design of the experiment is similar to the set up described by Merkouriadi et al.46. Where available, we force the model with N-ICE2015 observations. At other times, we use the ERA-Interim reanalysis83. An earlier comprehensive evaluation of ERA-Interim for the N-ICE2015 winter found good agreement between the reanalysis and observations5. For the period before the field campaign we apply the reanalysis along the satellite derived back trajectory54 of the ice floe from Drift 1. We apply a constant ocean heat flux of 2.7 W m−2 throughout the simulation . We prescribe an initial ice thickness on 1st September of 1.2 m, and snow depth of 0 cm. The initial ice temperature is assumed to decrease linearly from −0.25 °C at the ice top to −1.9 °C at the ice bottom. For further details of the experiment set up see Merkouriadi et al.46.We apply the 1-D thermodynamic sea ice model, HIGHTSI91. These changes did not alter any conclusions drawn.We conduct two idealised experiments to explore the impact of changes in precipitation on sea ice mass balance Fig. . In the et al.57 derived the mean daily sea ice concentration around the ice camp from the AMSR2 microwave radiometer92 , centred on the camp57.Fransson r92 Fig. . These d93 and Doulgeris94  synthetic aperture radar images to calculate the lead fraction around the N-ICE2015 camp. We process the images to 40 meters ground resolution and segment these using the method from Doulgeris and Eltofts94 Fig. . We subset al.42. Rösel et al.42 measured the total thickness of snow and sea ice by electro-magnetic induction  on a large scale. From this, they calculated the ice thickness by subtracting the snow depth measured with a snow probe 42.We estimate the volume of new ice growth in leads opened by storms using the areal fraction of leads calculated in the segmented images, as well as ice thickness measurements taken within these leads on 12 and 14 February (on average 0.28 m thick). Lead ice thickness surveys were conducted by drilling. We assume an average sea ice thickness of existing snow-covered ice of 1.0–1.5 m, based on extensive ice thickness measurements from Rösel 95 shown in Fig. et al.59, and followed standard methods reported by McPhee et al.91.Ocean-to-ice heat fluxes−2. Storm periods are based on Cohen et al.34, and are highlighted in Fig. −2. Mixing in the ocean is often still enhanced a few days after a storm  during the winter storms to be 6.7 W morm Fig. . Therefoet al.60 using microstructure profile observations96. Typically, 3 sets of 3 microstructure profiles were conducted daily between 0800 UTC and 2000 UTC, using two loosely tethered free-fall MSS-90 microstructure profilers97 developed by ISW Wassermesstechnik. Each final set of microstructure profiles is therefore the result of 3 combined individual microstructure profiles. These sets were used to estimate the profiles of ocean heat fluxes presented in Fig. et al.98. The processed data provide 0.2 m averaged temperature and salinity measurements and 1 m averaged dissipation rates. The mixed layer depth shown in Fig. et al.62.Dissipation rates, mixed layer depth, and ocean heat fluxes at the pycnocline depth for the N-ICE2015 winter, shown in Fig. fCO2 observations99 shown in Fig. et al.57. The data were obtained by infrared analysis of equilibrator headspace samples. The seawater was supplied from an intake located midships, at approximately 5 m water depth. The measurements were made using an instrument supplied by General Oceanics and designed following the principles presented by Pierrot et al.100 using two-stage showerhead equilibration and a LICOR 7000 nondispersive infrared detector57.The 83. Specifically, we show the sea ice extent before and after each of the six N-ICE2015 storms. Together with this we show the 10 m wind speed and mean sea level pressure field averaged over the duration of each storm  and the ERA-Interim atmospheric reanalysisorm Fig. . The dir−3, and use the specific latent heat of fusion of pure ice of 333500 J kg−1 multiplied by 0.89, following Bitz and Lipscomb101.Using the OSI-SAF sea ice drift product with a 62.5 km resolution, we calculate the import/export of sea ice area into a box covering the Atlantic Water inflow region north of Svalbard, throughout the N-ICE2015 winter Fig. . We defi102; this adds further uncertainty to the ice-melt estimate.Caution should be taken with these heat flux estimates, because we do not account for ice lost due to convergence (e.g. ridging or rafting). However, we expect this term to be small compared to the ice-melt term. For comparison, ice export from the box contributed a maximum of 25% of the total area loss. Our method also does not account the melt of ice floes that only partially melt from below (e.g. Fig. Supplementary Info


Therefore, peptides hydrolyzed from soy protein are promising natural ingredients for nutraceutical applications assisting in the management of diabetes.In this study, the peptides of soy protein obtained by enzymatic digestion with proteases were analyzed for their antidiabetic, antihypertensive, and antioxidant activities. Peptides prepared with alkaline proteinase (AP) exhibited the highest α‐glucosidase inhibitory activity compared with those from papain and trypsin digestion. AP hydrolysates also exhibited dipeptidyl peptidase IV (DPP‐IV) inhibitory, angiotensin‐converting enzyme (ACE) inhibitory, and antioxidant activities. Gastrointestinal digestion of peptides enhanced α‐glucosidase, DPP‐IV, and ACE inhibitory activities compared with AP hydrolysates. AP peptides showing highest α‐glucosidase inhibitory activity were purified by anion‐exchange and size‐exclusion chromatography， and identified using tandem MS. We found three novel α‐glucosidase inhibitory peptides with sequences LLPLPVLK, SWLRL, and WLRL with IC DM presents in two main forms: Type 1 insulin‐dependent DM (T1DM) constitute 10% of all DM cases, and type 2 non‐insulin‐dependent DM (T2DM) constitute 90% of all DM cases. It is predicted that the number of patients with diabetes will increase from 350 million today to 592 million by 2035  inhibitory activity of the soy protein hydrolysates. The potential of soy bioactive peptides as ingredients for nutraceutical or functional food formulation is discussed.22.1Saccharomyces cerevisiae, acarbose hydrate; dipeptidyl peptidase IV (DPP‐IV) from human; gly‐pro‐p‐nitroanilide, hippuryl‐l‐histidyl‐l‐leucine (HHL), angiotensin I‐converting enzyme (ACE) from rabbit lung (2.0 U/mg); and hippuric acid (HA), DEAE‐52, and gel Sephadex G‐15 were obtained from Sigma‐Aldrich. All other chemicals and reagents were of analytical grade and commercially available.Soy protein powder was purchased from Harbin Gaoke Group Co., Ltd. Enzymes including alkaline proteinase (200 U/mg), papain (800 U/mg), trypsin from porcine pancreas (250 U/mg), pepsin from porcine gastric mucosa (500 U/mg), and pancreatin from porcine pancreas were purchased from the Summus Chemical Company; 4‐nitrophenyl‐α‐D‐glucopyranoside (PNPG), α‐glucosidase from 2.2g, 10 min), it was collected supernatant and stored at −20°C until use. The degrees of hydrolysis (DH) values were measured by “pH‐stat” method described by Adler‐Nissen . Subsequently, the protein slurry was heated to 100°C for 10 min for denaturation. After cooling down, 0.5 mol/L sodium hydroxide (NaOH) and 0.5 mol/L hydrochloric acid (HCl) aqueous solutions were used to adjust the pH of the slurry to the optimum conditions recommended by the manufacturers for each protease: AP , papain , and trypsin . The protease  was added to initiate the protein enzymatic hydrolysis. Temperature and pH values were kept constant until the degree of hydrolysis no longer changed. Then, the hydrolysates were heated in a boiling water bath for 10 min to inactive the protease. After removal of the precipitate by centrifugation  on a TSKgel 2000 SWxl column (7.8 mm × 300 mm). One hundred milligram of hydrolysate powder was dissolved in 10 ml mobile phase , sonicated for 5 min, centrifuged at 8,000 rpm for 10 min, and then filtered through the microporous membrane. The injection quantity was 10 μl with the flow rate of 0.5 ml/min. The effluent was monitored by UV detector at 220 nm. Cytochrome C , bacitracin , leupeptin (MW: 427 Da), and tryptophan (MW: 204 Da) were used as molecular weight standards.2.42CO3 solution. The absorbance of released product PNP was measured immediately at 405 nm. Sodium phosphate buffer and acarbose (10 mg/ml of the buffer) were used as the negative and positive control, respectively. The percentage of α‐glucosidase inhibition was calculated as follows:A is the absorbance of the supernatant in which the sample is replaced by the same amount of buffer; ODS is the absorbance of the supernatant with the tested sample; and ODBis the absorbance of the supernatant in which the tested sample and α‐glucosidase solution are replaced by the same amount of buffer. IC50 value was obtained and used to evaluate the α‐glucosidase inhibition rate of the sample, which was calculated as the concentration of the tested sample required for α‐glucosidase inhibition rate of 50%.α‐glucosidase inhibitory activity assay was performed according to a method by Kim with slight modifications  at 37°C for 10 min, after added 50 μl of DPP‐IV ; the mixture was incubated at 37°C for 30 min; and then, 100 μl acetic acid–sodium acetate (1 M) was added to terminate the reaction. The DPP‐IV inhibition rate (%) was calculated as following:A is the absorbance of the supernatant in which the tested sample was replaced by the same amount of buffer; ODS is the absorbance of the supernatant with the tested sample; ODB is the absorbance of the supernatant in which the tested sample and DPP‐IV solution are replaced by the same amount of buffer; and ODN is the absorbance of the supernatant in which the DPP‐IV solution is replaced by the same amount of buffer.DPP‐IV inhibition assay was according to the method from Harnedy et al with minor modifications  using an elution of formic acid (0.4%): methanol (3:7 v/v) at a constant flow rate of 0.5 ml/min, and HA was detected by UV detector at 228 nm. The ACE inhibition rate (%) was calculated as follows:A is the chromatography peak area of the supernatant in which the tested sample is replaced by the same amount of buffer; C is the chromatography peak area of the supernatant with tested sample; and B is the chromatography peak area of the supernatant with no tested sample and ACE solution.The determination of ACE inhibitory activity was performed by using in vitro method described by Wu and Ding with slight modifications  were added respectively and mixed well, with the addition of 1 ml of 8.8 µmol/L H2O2 (0.03%) reaction initiated. The mixture was incubated in a 37℃ water bath for 30 min and then centrifuged at 2,896 g for 5 min to remove the precipitation. The absorbance of the supernatant was measured at 510 nm. The hydroxyl radical clearance rate was calculated as follows:A1 is the absorbance of the sample supernatant, A2 is the absorbance of the supernatant in which the salicylic acid–ethanol is replaced by the same amount of ethanol, and A0 is the absorbance of the supernatant in which the sample is replaced by distilled water. The ascorbic acid was used as a positive control.The antioxidant activity of the soy protein peptides was determined by a hydroxyl radical scavenging assay involving in the Fenton reaction, according to Hanasaki . FeSO4 aqueous solution was used as positive control.The FRAP assay was used to determine the antioxidative of the samples using the method described by Tsai et al with some modifications  pre‐equilibrated with the starting buffer . The adsorbed peptides were eluted as the concentrations of NaCl increased  in the starting buffer with a flow rate of 1.5 ml/min for 6 hr. The elution peaks were monitored at 220 nm, and the fractions were collected every 5 min. Factions eluted within each peak were pooled together, desalted with dialysis bag (200 Da cutoff), and assessed for their α‐glucosidase inhibitory activities. The separation process was repeated for gathering enough to further purification. Then, the sample was concentrated by rotary evaporation and purified by the size‐exclusion chromatography for further analysis.2.9.21‐1 was concentrated by rotary evaporation and subjected to the subsequent analysis and identification procedure.Three microliters of fraction from ion‐exchange chromatography (10 mg/ml) was loaded into a chromatography column (6 mm × 60 cm) packed with the pretreated gel Sephadex G‐15 . Then, the sample eluted using distilled water with the flow rate of 0.5 ml/min. The peptide elution was monitored by measurement of the absorbance at 220 nm, and fractions were collected. Separation process was repeated multiple times, and the α‐glucosidase inhibitory activities of collected fractions were analyzed. The fraction with the α‐glucosidase inhibitory activity marked by H2.101‐2were analyzed by  with online nanospray LC‐MS/MS on a Q Exactive Plus coupled to an EASY‐nano‐LC 1200 system (Thermo Fisher Scientific). Five microliters of the sample was loaded into the trap column , with a flow rate of 10 μL/min, and subsequently separated on the analytical column , with a linear gradient, from 3% to 38% B  in 60 min. The column flow rate was maintained at 0.5 ml/min with the column temperature of 40°C. The peptide sequences of the major peaks were performed by processing the ion series in MS/MS spectra using Peaks Studio 8.5 and manual interpretation.The amino acid sequence and the molecular mass of HA spray voltage of 2 kV was applied, and the instrument, Q Exactive Plus, was operated under switching automatically between MS full scan and data‐dependent fragmentation modes with 27 of collision energy. Ions were scanned at high resolution , and the MS scan range was 100–1500 m/z at both MS1 and MS2 levels.2.11Synthetic peptides were provided by Qingdao China Peptides Co., Ltd. using the conventional Fmoc solid‐phase synthesis method. Synthetic peptides were purified and determined by HPLC‐MS analysis with the purification rate over than 98%.2.12SD). Statistical calculation was performed by ANOVA with IBM SPSS statistics, version 25 (IBM Inc.). Differences were considered to be significant at p < 0.05.The data were expressed as the mean of three replicates and standard deviation  was hydrolyzed with AP, papain, and trypsin, respectively, and α‐glucosidase inhibitory activity of hydrolysates was analyzed.As shown in Figure Table 3.250 = 0.52 ± 0.05 mg/ml) was higher than the hydrolysates that the IC50 of α‐glucosidase inhibitory activity was 4.94 ± 0.07 mg/ml.The soy protein peptides obtained by AP hydrolysis were freeze‐dried after the removal of insoluble precipitation. Inhibitory activities against α‐glucosidase and DPP‐IV of soy protein peptides were measured. α‐Glucosidase is a membrane‐bound enzyme located in the epithelium of the small intestine that catalyses the cleavage of glucose from disaccharides , while it was weaker than that of vitamin C (IC50 = 0.54 ± 0.04 mg/ml). As shown in Figure 4 solution was linearly related to concentration, and at the same concentration, ferrous sulfate possessed stronger reduction ability.Since oxidative stress also affects the development of diabetes, the antioxidative activities of hydrolysate were evaluated. The soy hydrolysates obtained by AP exhibited hydroxyl radical scavenging ability , and after intestinal digestion, it reached to 77.64 ± 1.07%. For inhibitory activities against DPP‐IV, compared with undigested soy peptides, we also observed significant change (p < 0.05) after gastric and intestinal digestion, and it was 44.51 ± 0.95% and 47.94 ± 1.10%, respectively. For ACE inhibition activity, we found that after gastric digestion, it increased slightly to 21.71 ± 1.52% (p > 0.05), and after intestinal digestion, the inhibition rate enhanced significantly to 51.43 ± 1.74% (p < 0.05). Akillioglu and Karakaya reported an increase of ACE inhibitory activity in legume hydrolysates after the gastrointestinal digestion  were obtained after gradient elution through DEAE‐52. α‐glucosidase inhibition rates of each fraction were determined, and the results were shown in Figure 1 was washed off by distilled water, it was found that H1 was only the effective inhibitor on the α‐glucosidase with inhibition rate 87.10 ± 2.70%, and the inhibitory activity was higher than the original hydrolysates. All other fractions were not able to inhibit α‐glucosidase.In order to purify peptides with α‐glucosidase inhibitory activity, successive chromatographic separations were performed with anion‐exchange resin DEAE‐52 and Sephadex‐15 gel. As shown in Figure 1 fraction by rotary evaporation, then purified further by size‐exclusion chromatography. As shown in Figure 1fraction revealed a relatively clear molecular weight distribution profiles with only two main peaks . Figure 1‐2 showed high α‐glucosidase inhibition activity up to 95.35 ± 2.70% inhibitory rate, which was much higher than H1‐1 with 26.08 ± 2.12% inhibitory rate.We collected and concentrated H3.61‐2with EASY‐MS. The results showed four abundant ions with m/z of 893.6, 588.4, 675.4, and 745.4, which revealed four oligopeptides that would be further sequenced using LC‐MS/MS. We obtained the complete amino acid sequences by amino acid composition analysis and manual calculation of the MS/MS spectrum. As shown in Figure Aspergillus oryzae) (Kang, Yi, & Lee, We determined molecular weight of the purified fraction H3.750 value of 237.43 ± 0.52, 182.05 ± 0.74, and 165.29 ± 0.74 μmol/L. It has been suggested that the inhibitory mechanism could be through the hydrophobic interactions of the polypeptide with the active site of the enzyme, as it has been reported for other inhibitors (Bharatham, Bharatham, Park, & Lee, The synthesized peptides have been confirmed the great inhibitory effect on α‐glucosidase activity. The hexapeptides, MLPVMR, did not show inhibitory activity. However, other three peptides, LLPLPVLK, SWLRL, and WLRL, had great inhibitory influence on α‐glucosidase with the IC450 α‐glucosidase inhibitory activity values of LLPLPVLK， SWLRL, and WLRL were 237.43 ± 0.52, 182.05 ± 0.74, and 165.29 ± 0.74 μmol/L, respectively. Compared with other α‐glucosidase inhibitory peptides, these three peptides we found showed different structural homology from them, as well as lower IC50 value than acarbose. Therefore, this research can be referenced for the development of novel antidiabetic peptide nutraceuticals. Also, in order to validate the findings of the present study, further research in cell culture and in vivo should be needed.In summary, the peptides possessing high α‐glucosidase inhibitory effect were successfully prepared from soy protein treated via an AP process at the DH value of 36.84 ± 1.20% in the present study. The soy protein peptides also exhibited antioxidative activity and inhibition activities against DPP‐IV and ACE. The peptides with α‐glucosidase inhibition activity were separated with anion‐exchange resin, Sephadex G‐15 gel, which was determined using LC‐MS/MS sequentially. The ICThe authors declare that they do not have any conflict of interests.Human testing and animal testing were unnecessary in this study.


Mycobacterium tuberculosis and Plasmodium falciparum. Many NDH-2 inhibitors have been identified, but rational drug development is impeded by the lack of information regarding their mode of action and associated inhibitor-bound NDH-2 structure. We have determined the crystal structure of NDH-2 complexed with a quinolone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). HQNO is nested into the slot-shaped tunnel of the Q-site, in which the quinone-head group is clamped by Q317 and I379 residues, and hydrogen-bonds to FAD. The interaction of HQNO with bacterial NDH-2 is very similar to the native substrate ubiquinone (UQ1) interactions in the yeast Ndi1–UQ1 complex structure, suggesting a conserved mechanism for quinone binding. Further, the structural analysis provided insight how modifications of quinolone scaffolds improve potency (e.g. quinolinyl pyrimidine derivatives) and suggests unexplored target space for the rational design of new NDH-2 inhibitors.Type II NADH:quinone oxidoreductase (NDH-2) is a proposed drug-target of major pathogenic microorganisms such as   •First crystal structure of bacterial NDH-2 inhibited with the quinolone inhibitor HQNO•HQNO is a competitive inhibitor of quinone and targets the Q-site of NDH-2.•A conserved quinone binding mechanism among bacterial and yeast NDH-2 suggested•Rational design of new NDH-2 quinolone scaffold inhibitors suggested Unlike the large multi-subunit complex of the proton pumping type I NADH:quinone oxidoreducatase (complex I) .,13.ndh-2 potency . A numbeeveloped .Fig. 1Qu50 of 122 nM. This suggests the primary target of RYL-552 might be at the Q-site. Here, we present the crystal structure of the NDH-2–HQNO complex at 2.8 Å resolution. The NDH-2 inhibited structure reveals HQNO bound at the Q-site of NDH-2 and its inhibition mechanism competitive against quinone substrates. Further structural analysis shows a molecular framework for understanding both the binding of physiological quinone substrates and competitive inhibitors for rational drug development targeting NDH-2.Although many NDH-2 inhibitors have been identified, rational drug design is impeded by the lack of information regarding their modes of action and the unsolved inhibitor-bound structure. Defining the inhibition mechanism of quinolones using conventional inhibition kinetics has proven difficult, and results have suggested both competitive and non-competitive inhibition ,22,24,2522.1C. thermarum NDH-2 derivatives were expressed and purified as described previously [Wild-type (WT) and I379E eviously ,8.2.2−1 cm−1). For the HQNO inhibitory assay final NADH and menadione (MD) substrate concentrations were fixed at 200 and 50 μM, or at 200 and 400 μM, respectively. HQNO concentrations were varied from 0 to 100 μM and 0 to 300 μM for WT and I379E variants respectively to determine IC50 values. Enzyme concentrations used were typically 13.5 and 60.0 ng mL−1 for the WT, and I379E variants respectively. Each reaction mix was pre-incubated with MD and HQNO for 2 min and the reaction was initiated by adding NADH to the mix. The activity was normalised against a control sample with no HQNO present in the assay mix. Activity assay at each HQNO concentration was performed in triplicate. For the inhibitory assay using a quinolinyl pyrimidine compound final NADH and MD substrate concentrations were fixed at 200 and 50 μM, respectively. Enzyme concentration used was typically 15.0 ng mL−1. The compound concentrations tested were 0, 10 and 50 μM, respectively.NADH:menadione oxidoreduction assay was performed at 37 °C in 50 mM Tris-HCl buffer pH 8.0 containing 150 mM NaCl, 1% dimethyl sulfoxide and 1% octylglucoside as previously described . Activit2.3D, l-lysine, 4% (v/v) dimethyl sulfoxide and 1 mM 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Crystals were harvested on day four, flash-frozen and stored in liquid nitrogen. NDH-2–HQNO co-crystal diffraction data were collected at the Australian Synchrotron MX2 beam-line equipped with an ADSC Quantum 315r detector with 30% beam attenuation, 1 s exposures and 1° oscillation angle. Data were processed using XDS [5WED). The structures were refined using PHENIX [Crystallisation was performed employing the hanging-drop vapour diffusion method at 18 °C as previously described . NDH-2–Hsing XDS , Moleculsing XDS  with a Wg PHENIX  with NCSg PHENIX  was usedg PHENIX  to creat2.45JWC) was used for docking. The crystal structure of NDH2 was prepared using Protein Preparation Wizard [−1 of the best-modelled pose.The structures of RYL552 and Triton X100 were built in Maestro  and thenn Wizard ,40. RYL5n Wizard . The cen2.5Co-ordinates and structure factors have been deposited in the Protein Data Bank with an accession number 6BDO.33.1C. thermarum NDH-2 variant, which has significantly reduced quinone-binding affinity (KMMD 20-fold > wild-type), but retains maximal NADH oxidation activity [C. thermarum NDH-2. We determined the HQNO inhibition activity against the NDH-2 derivatives using menadione (MD) at excess (over ten times the KM) and low (close to the KM) concentrations. We expected to observe an inverse correlation between the degree of HQNO inhibition activity and the MD concentration if HQNO and MD directly competed for binding. WT NDH-2 had a IC50 value of 10.5 ± 1.3 μM HQNO in the presence of 400 μM MD  was observed with >50 μM HQNO . The exception was a D380 side chain carboxyl, which moved away from the Q-site . In addition, a hydrophobic side chain of M485 holds the opposite side of the quinone aromatic ring, although M485 is not an equivalent residue to I379 of C. thermarum NDH-2 . Nonetheless, the packing orientation of the quinone-head groups in both structures was highly comparable ) were reported for the yeast Ndi1 [1. They could have also arisen from an endogenous UQ6 bound in the purified Ndi1 [1 structure [Our bacterial NDH-2–HQNO structure suggests the inhibition mechanism of HQNO is to block the quinone substrate accessing to the FAD isoalloxzine. Furthermore, together with ICast Ndi1 . These mied Ndi1  and the tructure . Further3.4T. gondii TgNDH2-I [TgNDH2-I.Although a number of quinolone derivatives have been developed, the structural basis for understanding how modifications of quinolone scaffolds improve potency is lacking. Thus, we analysed the NDH-2–HQNO and the Ndi1–UQ structures to gain insight into the molecular basis of the potent inhibition mechanisms of these compounds. In the Ndi1 structure, the ubiquinone isoprenyl tail is accommodated in the hydrophobic groove  of the C-terminal membrane-anchoring domain A. In ourTgNDH2-I , suggestP. falciparum PfNDH-2 and M. tuberculosis Mtb NDH-2 have been successfully developed [C. thermarum NDH-2, the slightly deeper hydrophobic pocket is formed with Y13, T46, and A316 residues  of the C-terminal membrane anchoring domain. This result supports our hypothesis that the main inhibition mechanism of the PfNDH-2 inhibitor RY-552 is blocking of the Q-site. Notably, the tetramethylbutyl phenyl group of Triton X-100 contacts the same hydrophobic groove . In addition, a number of unmodelled large disordered electron densities, which are potentially of the detergent, were observed. Similarly, many Triton X-100 have been modelled in yeast Ndi1 structures , and the authors have performed co-crystallisation in the presence of 0.5 mM UQ to obtain Ndi1–UQ complex structures. Therefore, highly-concentrated Triton X-100 and inhibitors might compete for binding at the Q-site of PfNDH-2.Our analysis suggests that the primary target of the PfNDH-2 inhibitor RYL-552 is the Q-site, given that mutations introduced in the proposed allosteric inhibition sites did not eliminate the inhibition activity of RYL-552 . In addin silico A and B, e Q-site C and D wng model C and D. c groove A and B. 4via a direct hydrogen-bonding interaction between FAD and quinone molecules among NDH-2 enzymes. The NDH-2–HQNO structure provides insight into the molecular mechanism of inhibition for new NDH-2 inhibitors (e.g. quinolone and quinolinyl pyrimidine derivatives) and suggests unexplored target space (e.g. the hydrophobic cavity extending away from the quinone-binding tunnel) for the rational design of new NDH-2 inhibitors. Note that during the peer-review process of this article, another group reported crystal structures of Ndi1 with three Q-site inhibitors  [In conclusion, the structural analysis of bacterial NDH-2 inhibited by HQNO provides molecular details of the interactions and residues involved in quinone binding. The interaction of HQNO with bacterial NDH-2 is very similar to the native substrate UQ interactions in the yeast Ndi1–UQ complex structure suggesting a conserved mechanism for quinone binding, and moreover, a common hydride transfer catalytic mechanism mediated nd 5YJY) .The side chain of D380 changes position (cyan to green) upon HQNO binding. The structures of wild-type NDH-2 (wheat) and the HQNO complex (white) are overlaid.Fig. S1The following is the supplementary data related to this article.in silico docking studies. E.J.P., G.M.C. and Y.N. analysed data and wrote the manuscript with help from other authors; E.J.P., G.M.C and Y.N. designed the research and directed the project.J.P., Y.S., H.R.B., E.R.R. and Y.N. expressed and purified the protein, and performed assays. D.A. and Y.N crystallised the protein and solved the crystal structure. W.J. performed the The authors declare no competing interests.Transparency document.Image 1


Bacillus subtilis possesses four lipoteichoic acid synthases LtaS, YfnI, YvgJ and YqgS involved in the synthesis of cell wall. The crystal structure of the extracellular domain of LtaS revealed a phosphorylated threonine and YfnI was identified in two independent phosphoproteome studies. Here, we show that the four LTA synthases can be phosphorylated in vitro by the Ser/Thr kinase PrkC. Phosphorylation neither affects the export/release of YfnI nor its substrate binding. However, we observed that a phosphomimetic form of YfnI was active whereas its phosphoablative form was inactive. The phenotypes of the strains deleted for prkC or prpC (coding for a phosphatase) are fairly similar to those of the strains producing the phosphoablative or phosphomimetic YfnI proteins. Clear evidence proving that PrkC phosphorylates YfnI in vivo is still missing but our data suggest that the activity of all LTA synthases may be regulated by phosphorylation. Nonetheless, their function is non-redundant in cell. Indeed, the deletion of either ltaS or yfnI gene could restore a normal growth and shape to a ΔyvcK mutant strain but this was not the case for yvgJ or yqgS. The synthesis of cell wall must then be highly regulated to guarantee correct morphogenesis whatever the growth conditions. LTA has been shown to play an important role in bacterial growth and physiology, cation homeostasis, morphogenesis and cell division7. Unlike Staphylococcus aureus which only has one LTA synthase (LtaS) that produces LTA8, four LtaS paralogues LtaS, YfnI, YvgJ and YqgS are present in Bacillus subtilis. Three of them are authentic synthases whereas YvgJ acts as a primase that adds the first glycerolphosphate subunit to the lipid anchor9. All enzymes are active in vitro. LtaS is described as the “house-keeping” enzyme necessary during vegetative growth, YfnI is assumed to be the “stress” enzyme important during cell envelope stresses and YqgS is important during sporulation. They are predicted to have five N-terminal transmembrane helices followed by a large extracellular enzymatic domain that is cleaved just after a conserved AXA motif by type I signal peptidases and released in the culture medium11. Proteomic studies showed that both extracellular domains of LtaS and YfnI (eLtaS and eYfnI) are found in B. subtilis late-exponential phase culture supernatants when grown in minimal medium12. Interestingly, in the absence of LtaS, YfnI becomes more efficient in LTA production and synthesizes polymers with increased length suggesting that YfnI activity is modulated by LtaS9. The four LTA paralogues, that possess a high percentage of sequence similarity, therefore seem to have interdependent activities and a partial functional redundancy in B. subtilis9.The bacterial cell wall is a crucial structure that protects the cell from external stresses or damages and confers shape and resistance to osmotic pressure. Lipoteichoic acid (LTA) is an important component of the cell wall in Gram-positive bacteria, which is linked to the membrane by a lipid anchor. The polymer is composed of a polyglycerolphosphate (poly(GroP)) backbone chain tethered to the membrane by a diglucosyl-diacylglycerol glycolipidB. subtilis14. Furthermore, in the eLtaS crystal structure the active site Thr297 was found to be modified by a phosphate group5. The replacement of this Thr by an Ala residue abolished LtaS activity. The authors suggested that it rather mimics an intermediate state than a phosphorylation by a kinase since this residue is located in a deep pocket but the donor for this phosphate group is unknown and remains an open question. In B. subtilis, several intracellular Ser/Thr protein kinases (STPK) have been characterized, including PrkC, PrkD and YabT. Among them, PrkC is the kinase most highly expressed during exponential and stationary growth phases15 when the LTA synthases are active. PrkC was found to be responsible for the phosphorylation of many proteins involved in different cellular processes including metabolism, cell division and morphogenesis20 and could be responsible for the phosphorylation of YfnI and LtaS. Interestingly, a link has also been established between one PrkC substrate, the YvcK protein involved in the maintenance of morphogenesis during growth on neoglucogenic carbon sources and the LTA synthase YfnI21. More specifically, the yfnI gene was identified in a transposon mutagenesis screen looking for ΔyvcK suppressive mutations; it was further demonstrated that deletion of yfnI in a ΔyvcK mutant could restore the growth of this strain on neoglucogenic carbon compounds. It has also been shown that phosphorylation of YvcK by PrkC is involved in B. subtilis morphogenesis. Namely, overproduction of YvcK could rescue the growth and shape defect of an ΔmreB mutant and phosphorylation of YvcK was necessary for this rescue19. Considering all the previous data, our hypothesis was that YfnI, and maybe some of the other LTA synthases, could also be regulated by phosphorylation by PrkC.In addition, YfnI and LtaS were found to be phosphorylated. YfnI has been identified as phosphorylated protein in two independent phosphoproteome studies on B. subtilis. We showed that the PrkC protein kinase is able to phosphorylate the four LTA synthases in vitro. Regarding YfnI, its phosphorylation neither affected its export across the cell membrane nor its release in the culture medium nor prevented binding to its substrate. However, we observed that its phosphoablative form is inactive in vivo whereas its phosphomimetic form is active. Altogether, these results suggest a regulatory role of phosphorylation of YfnI on its activity with a probable contribution of the kinase/phosphatase duo PrkC/PrpC in this phosphorylation event.We therefore investigated the potential phosphorylation of YfnI and its paralogues and looked for its regulatory role in LTA synthesis or in cell morphogenesis in B. subtilis LTA synthases possess a high degree of sequence similarity . Then we tested their phosphorylation using the purified catalytic domain of the Ser/Thr kinase PrkC (PrkCc) and radioactive ATP  and that the main phosphorylation site is the Thr297 since all radioactive signal was lost for the eYfnI-T297A variant (lane 2) whereas some residual phosphorylation signal was detected for eYfnI-S298A and eYfnI-T303A (lanes 3 and 4). We also checked if eYfnI-P could be dephosphorylated by PrpC, the phosphatase associated with PrkC and we found that eYfnI-P was indeed dephosphorylated by PrpC  and that the presence of a phosphoryl group on the active center of the enzyme would inhibit LtaS activity. They also suggested that Thr297 phosphorylation rather mimics an intermediate state in the formation of poly(GroP) than a phosphorylation by a kinase since this residue is located in a deep pocket5. Despite its buried localization in the structure, our results indicate that this Thr297 is accessible for a phosphorylation by the protein kinase PrkCc. Furthermore, since PrkCc was able to phosphorylate eLtaS, eYfnI, eYvgJ and eYqgS in vitro, we can consider a possible common regulatory mechanism for the four B. subtilis LTA synthases.In the paper describing the 3D-structure of eLtaS, the authors showed that the replacement of the Thr297 by an Ala residue abolished LtaS functionin vivo by the B. subtilis PrkC kinase, it would need to take place in the cytoplasm of the cell before the export of the extracellular domain of the LTA synthases. The folding of the proteins is probably not fully complete allowing access to the future active sites. Consequently, the catalytic domains of the four LTA synthases should interact with that of PrkC. To get further evidence that these interactions might take place in the bacterial cell and that PrkC could phosphorylate YfnI  in vivo, we produced plasmids  or an unphosphorylated protein (YfnI-T297A-FLAG) . This result indicates that there is enough room in the catalytic site to receive the GroP molecule even when the Thr297 is phosphorylated or replaced by another residue. Recently, the structure of eLtaS from Listeria monocytogenes has been solved27 showing a phosphorylated catalytic threonine (Thr307Lm = Thr297Bs). Although the authors suggest that this phosphorylation is not physiological, they characterized two functional GroP binding sites in the protein that allows the simultaneous occupation of the phosphorylation of Thr307 and a GroP molecule in each of the sites within a compatible distance to permit the enzymatic catalysis. This second binding pocket has been identified in eLtaS from B. subtilis as well but a significant conformational change has to occur before the catalytic reaction27. Furthermore, LTA synthases belong to the alkaline phosphatase and arylsulfatase superfamily for which Ser and Thr residues are often phosphorylated to be active28 suggesting that a similar mechanism may be necessary for LtaS and for the other LTA synthases in B. subtilis.In order to analyze the role of this phosphorylation on YfnI properties, we first tested if it affects the affinity of the enzyme for the substrate mimic, GroP. For this purpose, the unphosphorylated protein, a phosphorylated protein preparation (eYfnI was beforehand incubated with PrkCc), and the inactive eYfnI-T297A) or potential phosphomimetic (eYfnI-T297D) forms of the protein were incubated with GroP then analyzed by isothermal shift assays. Surprisingly, we found that the phosphorylated or modified forms of eYfnI were all able to bind the substrate with only a slightly smaller affinity  and the presence of a phosphoryl group on the active center of the enzyme would inhibit LtaS activity5. In a ΔltaS strain, we observed that neither the deletion of prpC or prkC, nor the overproduction of GFP-PrpC had an effect on the synthesis of the longer LTA chains  strains had a WT shape as expected. We found that only the absence of ltaS or yfnI was able to complement the morphological phenotype of the ΔyvcK mutant whereas the absence of one of the other two enzymes, YvgJ or YqgS, had no effect. These results confirm that the activities of the LTA synthases in vivo are not perfectly redundant, consistent with previously described results9.To analyze the possible participation of PrkC and PrpC in the regulation of YfnI activity ain Fig. . We alsoyse Fig. . The sineen Fig. . All sinin vivo, we then used the same morphological screen to test the activity of phosphomimetic and phosphoablative forms of YfnI and for this, constructed strains expressing yfnI-T297A or yfnI-T297D in a ΔyvcK genetic background  whereas the single mutant ΔyvcK, used as positive control, presented a width of 0.150 µm. The cell width data collected were consistent with the shape of bacteria on the images but also showed that cells of the single ΔprkC mutant were already a bit wider (0.116 µm) than WT cells (0.102 µm), which could explain why the cell shape of the ΔyvcK ΔprkC deletion strain was not completely restored. Furthermore, the size of the cells was significantly increased for strain ΔyvcK ΔprkC yfnI-T297D (0.152 µm) compared to strain ΔyvcK ΔprkC (0.140 µm) and reduced for the strain ΔyvcK ΔprpC yfnI-T297A (0.138 µm) compared to strain ΔyvcK ΔprpC (0.171 µm)  Fig. .Figure 6in vivo through its phosphorylation state that can be at least partially mediated by the PrkC/ PrpC kinase and phosphatase proteins. It is also important to remember that PrkC and PrpC have multiple substrates in the cell involved in several cellular processes. Therefore, deletion of prkC and prpC may have quite pleotropic effects explaining the small differences in cell size observed here. However, our results suggest that PrkC could phosphorylate YfnI in vivo but it may be not the only kinase responsible for this phosphorylation event.In conclusion, these results suggest that there might be a regulation of YfnI activity B. subtilis and E. coli were used. All the strains and plasmids used in this study are listed in Table Standard procedures for molecular cloning and cell transformation of yfnI was amplified by PCR using specific primers and inserted between ApaI and EcoRI sites in the pSG1154 plasmid where the gfp gene has been replaced by a sequence encoding a FLAG peptide.For the generation of FLAG-fusion proteins, yfnI-flag to generate fusion proteins with phosphoablative (T297A) or phosphomimetic (T297D) replacements; as well as on plasmid pProEX-eyfnI to produce N-terminal 6His-tagged extracellular domains of YfnI carrying the mutations S298A, T303A and the phosphoablative (T297A) or phosphomimetic (T297D) replacements.Site directed mutagenesis was then carried out on the resulting plasmid pSG1154-yvgJ and eyqgS were amplified by PCR using specific primers and used to replace eyfnI in the pProEX-eyfnI plasmid between the BamHI and XbaI sites.To produce the extracellular domains of YvgJ and YqgS with an N-terminal 6His-tag, eyfnI  or ltaS  were amplified from chromosomal DNA by PCR with two pairs of primers containing overlapping regions. The two above described fragments and a fragment containing the kanamycin or the chloramphenicol resistance genes respectively were then joined by isothermal assembly using the Gibson Assembly® Master Mix from NEW ENGLAND BioLabs to generate the whole sequences yetO-kan-yfnHG and nagB-cat-yflDA and the resulting products were used to transform the WT168 strain to generate the strains SG445 and SG600 (Table The flanking sequences of yfnI-flag and derived plasmids were used to amplify the modified yfnI genes. The flanking sequences of yfnI (yetO and yfnHG genes) were amplified from the chromosomal DNA by PCR with two pairs of primers containing overlapping regions. A global PCR using the three above described fragments and a fragment containing the kanamycin resistance gene were then used to amplify the whole sequence yetO-yfnI (WT or T297A or T297D)-kan-yfnHG and the resulting DNA were used to transform the BKE07260 strain to generate the strains SG394, SG399 and SG397  as previously described29 for eYfnI, eYfnI-T297A, eYfnI-T297D, eYfnI-S298A, eYfnI-T303A, eLtaS, eYvgJ and eYqgS. The tag was then removed by proteolytic digestion with the acTEV protease (10 units for 500 µg of protein during 90 min at 30 °C) and separated from the 6His-tag by a second purification step on Ni2+-NTA resin from which the proteins were obtained in the first wash step.pProEX-derived plasmids allowing the overexpression of N-terminal 6His-tagged extracellular domain of the LTA synthases were used to transform E. coli C41(DE3). Purification of 6His-tagged recombinant proteins was performed with Ni2+-NTA resin (Qiagen) as previously described in22 and30 respectively.Plasmids allowing the production of 6His-tagged catalytic domains of PrkC and YabT Table  were use2, 2 μg of MBP (Myelin Basic Protein) and 1 mM [γ-33P] ATP (1 μCi). MBP stimulates PrkC kinase activity and serves also as a phosphorylation control by being an exogenous protein kinases substrate24. For the dephosphorylation test of eYfnI, 2 μg of eYfnI were first phosphorylated as described above for 10 min at 37 °C (positive control) and when indicated 1 μg of PrpC was added and incubated for 10 additional minutes. The phosphorylation reaction was stopped by adding 5X SDS-sample buffer to the reaction mixtures before SDS-PAGE analysis. Gels were then dried and exposed to autoradiography.2 μg of eYfnI (or extracellular domains of the others LTA synthases) were incubated for 15 min at 37 °C with 2 μg of the catalytic domain of PrkC protein, PrkCc, (or YabT) in a 15 μl reaction mixture containing 10 mM HEPES, pH 8.0, 1 mM MgCl® Orange dye solution , in 40 mM NaCl, 1 mM MgCl2, 10 mM Tris/HCl, pH 8.0 and increasing concentrations of GroP (from 0 to 20 mM) and was heated from 25 °C to 65 °C in 0.5 °C steps. The fluorescence intensity (Ex/Em = 470/570 nm) of SYPRO® Orange was monitored using a real-time PCR apparatus CFX96 (Bio-Rad). The fluorescence of SYPRO® Orange dye changes when it interacts with the protein undergoing thermal unfolding. The denaturation temperature (Tm) was analyzed from the melt peak using CFX Manager software (Bio-Rad). The shift of Tm (ΔTm) induced by the presence of ligand was plotted against the concentration of ligand. Curve fitting was performed by using Microcal Origin 5.0 software with the Hill equation. To test the affinity of GroP for the phosphorylated form of eYfnI, 400 µg of the protein was first incubated with 50 µg of PrkCc in 10 mM HEPES, pH 8.0, 5 mM MgCl2 and 5 mM ATP for 60 min at 37 °C. eYfnI-P was then used as described above.In thin-walled 96-well PCR plates, each well (20 μl) contained 10 μM of eYfnI protein (or eYfnI mutated proteins) and 2 μl of the fluorescent SYPROB. subtilis strains were grown in LB or CE minimal medium supplemented with 0.5% gluconate as previously described in21.9.For LTA detection, cells were grown at 37 °C in 30 ml of CE minimal medium supplemented with 0.5% gluconate for 6 hours then treated as previously described600 = 1.2, then centrifuged for 10 min at 4000 rpm at 4 °C. Proteins from cell culture supernatants (SN) were precipitated by addition of 1/10th volume of TCA and incubated for 30 min at 4 °C. SN were centrifuged for 30 min at 4000 rpm and 4 °C then protein pellets were washed twice with acetone, left 5 min to dry then resuspended in 1/100th volume of Laemmli buffer. Crude extracts from cell pellets were obtained by resuspension in 1/30th volume of lysis buffer containing 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% NP40, 1 mM PMSF, 1 mM DTT, 25 U.ml−1 benzonase and 10 mg/ml lysozyme, and incubated for 30 min at 37 °C. 1/10th volume of 10% SDS and 1/2 volume of (2×) Laemmli buffer were added to the extracts. All protein extracts were heated at 100 °C for 10 min. Samples were run on a 12.5% SDS-PAGE and transferred to hybond-ECL membrane by electroblotting. The membrane was blocked with PBS solution containing 5% milk powder (w/v), for 3 hours at room temperature with shaking, then incubated with anti-FLAG antibodies diluted to 1/1000th overnight at 4 °C. After three washes, the secondary antibody, a peroxidase-conjugated Goat anti-Rabbit (Thermo scientific) antibody was used at 1/2000th dilution for one hour. After three washes, the membrane was incubated with ECL reagents (PerkinElmer) and scanned for chemiluminescence with an ImageQuant LAS4000 .For YfnI-FLAG detection, cells were grown at 37 °C in 30 ml of LB medium containing 0.5% xylose until ODStrains were grown for 6 h in CE minimal medium supplemented with 0.5% gluconate at 37 °C. Images were obtained by microscopy on a Zeiss Upright Axio Imager M2 microscope. Cell width was measured with the ObjectJ plugin of ImageJ software and a statistical analysis of these data was made using a Wilcoxon test in order to calculate p-values.32. The N-termini of YfnI, PrkCc and PrkCc-K40A were fused to the T18 or T25 catalytic domains of adenylate cyclase as described in19 and above. Co-transformed strains of E. coli BTH101 expressing several combinations of T18 and T25 vectors were plated on LB agar and incubated at 30 °C for 48 hours. One milliliter of LB medium, supplemented with 100 µg/ml of ampicillin, 50 µg/ml of chloramphenicol, and 0.5 mM of IPTG, was inoculated and incubated at 30 °C overnight. Ten microliters of the overnight culture were spotted on the LB medium plates containing appropriate antibiotics, 0.5 mM IPTG and 40 µg/ml Xgal. The plates were incubated at 30 °C overnight.The bacterial two-hybrid assay was performed as described inSupplementary information


Hypericum perforatum L., also known as Saint John’s Wort, has been well studied for its chemical composition and pharmacological activity. In this study, the antiviral activities of H. perforatum on infectious bronchitis virus (IBV) were evaluated in vitro and in vivo for the first time. The results of in vitro experiments confirmed that the antiviral component of H. perforatum was ethyl acetate extraction section (HPE), and results showed that treatment with HPE significantly reduced the relative messenger ribonucleic acid (mRNA) expression and virus titer of IBV, and reduced positive green immunofluorescence signal of IBV in chicken embryo kidney (CEK) cells. HPE treatment at doses of 480–120 mg/kg for 5 days, reduced IBV induced injury in the trachea and kidney, moreover, reduced the mRNA expression level of IBV in the trachea and kidney in vivo. The mRNA expression levels of IL-6, tumor necrosis factor alpha (TNF-α), and nuclear factor kappa beta (NF-κB) significantly decreased, but melanoma differentiation-associated protein 5 (MDA5), mitochondrial antiviral signaling gene, interferon alpha (IFN-α), and interferon beta (IFN-β) mRNA levels significantly increased in vitro and in vivo. Our findings demonstrated that HPE had significant anti-IBV effects in vitro and in vivo, respectively. In addition, it is possible owing to up-regulate mRNA expression of type I interferon through the MDA5 signaling pathway and down-regulate mRNA expression of IL-6 and TNF-α via the NF-κB signaling pathway. Moreover, the mainly active compositions of HPE analyzed by high-performance liquid chromatography/electrospray ionization–mass spectroscopy (ESI-MS) are hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, and a combination of these compounds could mediate the antiviral activities. This might accelerate our understanding of the antiviral effect of H. perforatum and provide new insights into the development of effective therapeutic strategies. The infectious bronchitis virus (IBV) is a prototype coronavirus containing a single-stranded positive-sense RNA genome . IBV is Hypericum perforatum L. belongs to the genus Guttiferae, which contains approximately 400 species all over the world. The extract of H. perforatum contains several active compounds, including flavonoids, naphthodianthrones, and phloroglucinol derivatives , and HIV  expression levels of IBV in CEK cells, tracheas, and kidneys were measured. The positive green immunofluorescence signal of IBV in CEKs was observed. In addition, hematoxylin-eosin (HE) staining of tracheas and kidneys was performed, and the relative mRNA expression of IL-6, tumor necrosis factor alpha (TNF-α), IFN-α, IFN-β, MDA5, MAVS, and nuclear factor kappa beta (NF-κB) in vitro and in vivo were analyzed. Finally, the antiviral principal chemical composition of H. perforatum extract was analyzed by high-performance liquid chromatography (HPLC)/ESI-MS. In summary, our findings for the first time showed that H. perforatum extract had significant antiviral effect on IBV, and it might up-regulate mRNA expression levels of type I interferon via MDA5 pathway and down-regulate mRNA expression levels of IL-6 and TNF-α through the NF-κB pathway. The HPE was found to be composed mainly of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, and a combination of these compounds could mediate the antiviral activities.In this study, we investigated the antiviral effects of The IBV M41 strain (GenBank: FJ904723.1) was provided by Key Laboratory for Laboratory Animals and Comparative Medicine, Northeast Agricultural University and propagated in 10-day-old specific pathogen-free (SPF) chicken embryos . 100 µl IBV M41 strain was inoculated into SPF chicken embryos under aseptic operation. The survival status of chicken embryos was observed every 12 h, and the chicken embryos that died within 24 h were abandoned. Then allantoic fluid from infected embryos was collected at 72 h post-inoculation and stored at −80°C.H. perforatum was picked from Tongjiang County, Sichuan Province  and authenticated by Professor Zhongbao Zhang . The reference specimen   chickens were purchased from Harbin Veterinary Research Institute, Heilongjiang Province, China. Experimental procedures were approved by the Institutional Animal Ethical Committee of Northeast Agricultural University (no. SRM-08). All animal studies were complied with the animal experiment guidelines of the Animal Experimentation Ethics Committee of Northeast Agricultural University.H. perforatum was prepared in the laboratory. In brief, the drug was pretreated with carbon dioxide supercritical extraction method, and the supercritical extraction extract (SEE) was obtained with a yield of 8.1% (wt/wt). Then, 15 g of the pretreated medicinal material was weighed, 1.0 L of 85% methanol was added  and the water layer (lower layer) were obtained. It was then extracted with ethyl acetate until the upper layer had no color. All ethyl acetate extractions and water extractions were combined and evaporated to obtain extract of H. perforatum ethyl acetate (HPE) and extract of H. perforatum water (HPW).Approximately 6 g of the crude extract of in vitro, the working solution of H. perforatum extract was diluted in M199 medium without serum to obtain a final concentration. The HPE was dissolved in 5% DMSO, while ribavirin (RT) was dissolved in physiological saline solution  in vivo experiments.A stock solution of HPE (200 mg/ml), HPW (200 mg/ml) and SEE (200 mg/ml) was prepared with dimethylsulfoxide , respectively. Immediately before each experiment 2. Then, IBV M41 strain was passaged in CEK cells for adaption. 500 µl IBV M41 allantoic fluid was inoculated into CEK cells in 5 ml cell culture flask, and the cytopathic effect (CPE) was observed. When the cells showed obvious typical CPE, the time and degree of the lesions were recorded and the first generation of IBV was collected by freezing and thawing cells repeatedly. Then, the collected first generation IBV was inoculated into CEK cells according to the above method, and the time and lesion degree of CPE were also recorded. The second generation IBV was collected according to the above method. After 10 times of passages of IBV in CEK cells, there appeared typical CPE of cell exfoliation and fusion at same time post inoculation regularly. IBV was collected and stored at −80°C until use. The results of adaptation and replication in CEK cells, such as CPE, reverse transcription polymerase chain reaction (RT-PCR), and IBV growth curve determined by tissue culture infective dose (TCID50) at different time were tested.CEK cells were primary cultured from 18-day-old SPF chicken embryo  and prepared according to the standard technique . The cel570 was determined. Using the mean values, the cell survival rate is calculated according to the following formula:The cytotoxicity test was carried out according to the published method of thiazolyl blue tetrazolium bromide (MTT), and minor modifications were made . The mon50 IBV and cultured at 37°C for 30 h. The cell samples were frozen and thawed repeatedly for three times, and the total RNA was extracted and reverse transcribed into complementary deoxyribonucleic acid (cDNA). The relative mRNA expression of IBV N gene was detected by real-time quantitative RT-PCR (qRT-PCR). In addition, according to the conventional method, the virus titer of cell samples was determined by TCID50. The specific operations were as follows: CEK cells were cultured in 96-well tissue culture plates, and when the cells were full of monolayer, IBV virus was diluted to 10−1, 10−2, 10−3…, and 10−12 times with serum-free M199, respectively. Then the monolayer CEK cells were washed with D-Hanks solution three times, and the D-Hanks solution was discarded. Then the 100 µl IBV virus diluent as mentioned above was added to each hole, and each virus diluent repeated eight holes. The cells were treated by the same method with M199 culture as normal control. The culture plate was cultured in 5% CO2 and 37°C for 48 h. The cytopathic effect (CPE) was observed every 24 h, and the number of CPE pores and no CPE pores were recorded. According to the Reed-Muench method as previously described   for 1 h, and then incubated with fluorescent labeled goat anti-rabbit IgG (1:500)  for 30 min in the dark. The fluorescent images were examined with a fluorescence microscope . At the same time, the infected cells treated with 10 μg/ml RT, and CEK cells infected with IBV, and the mock CEK cells were set as controls.50 of IBV M41 allantoic fluid per chick. Chicks in the control group were inoculated with 0.1 ml sterilized negative allantoic fluid in the same manner. The chicks of the three HPE groups and the RT group were orally administered varying doses of three HPE  and ribavirin (60 mg/kg), respectively. Both agents were administered twice daily for 5 days, beginning at 24 h after exposure to the virus. At the same time, the chicks of the IBV-infected group and the normal group were orally administered with PSS. At 2, 4, 6, and 8 days post-infection, six chicks in each group were bled before euthanasia and necropsy at each designed day. The trachea and kidney tissues were collected and immediately extracted to obtain the total RNAs. At the same time, tracheal and kidney in each group of chickens at 6 days postinfection were fixed in 10% buffer formaldehyde for histopathological assay.One hundred and forty four 2-week-old SPF chicks were randomly divided into HPE treated high dose group (HPE-TH), HPE treated middle dose group (HPE-TM), HPE treated low dose group (HPE-TL), the IBV infected group (IBV), the ribavirin treated (RT) group, and normal control (NC) group with 24 chicks in each group. Each group was completely isolated from the other groups, and housed in negative pressure-filtered air isolators under pathogen-free conditions. Chicks from in three HPE-treated groups, the IBV-infected group, and the RT group were inoculated by nasal drops with 0.1 ml of 100 EIDAfter washing in phosphate-buffered saline (PBS), the trachea and kidney tissues were fixed in 10% formaldehyde solution for 7 days, embedded in paraffin wax, cut into 3-μm-thick sections , and stained with HE using standard histological staining procedure. The slides were examined by light microscopy . The tissue slides were examined and evaluated according to the previous histopathologic scoring method for trachea  and kidnTreated cell samples were repeatedly frozen and thawed three times, and the equiponderant trachea and kidney tissues from each chick at various time points were prepared. Then, the total RNA was extracted using a Universal RNA Extraction Kit  according to the manufacturer’s instructions.All the concentration of RNA and the A260/A280 ratio were determined with NanoDrop 2000 Spectrophotometer , and the integrity of the extracted RNA was detected by agarose gel electrophoresis. The total RNAs were reverse transcribed into cDNAs by PCR instrument  for qRT-PCR, and the cDNAs were stored at −20°C.−ΔΔCt method . All primer pairs for real-time PCR detection are listed in t method .The amplification system was 2 μl cDNA, 0.6 μl forward and reverse primers, 10 μl universal SYBR Green (ROX) and 6.8 μl nuclease-free water, and the final volume of each system was 20 μl. The amplification reaction of qRT-PCR assays was conducted according to the following thermal profile: pre-incubation 1 cycles at 95°C for 600 s, followed by 2 step amplification: 42 cycles at 95°C for 15 s and 60°C for 30 s, followed by melting: 1 cycles at 95°C for 10 s, 65°C for 60 s, and 97°C for 1 s, followed by cooling 1 cycles at 37°C for 30 s.HPLC/diode array detector/ESI-MS analysis was performed on a Waters 2695 HPLC equipped with diode array detector and Waters Micromass ZQ ESI-electrospray  operating in positive and negative ion mode. According to relevant reports , the HPLThe operating conditions of the mass spectrometer were dry gas temperature, 350°C, flow rate, 50 L/min; nebulizer pressure, 30 psi; sheath gas temperature, 250°C, flow rate, 10 L/min; fragmenter voltage, 100 V; capillary voltage, 3,500 V; mass range, 50 –1,100 D.The sample solution was prepared by dissolving 100.0 mg dried HPE in 25 ml mobile phase and filtered by 0.22 μm filter before HPLC analysis. The content of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin in the HPE was determined by HPLC instrument and the chromatographic conditions described above. In the 25 ml analytical solvent, the hyperoside standard of 2.5 mg, or the quercitrin standard of 5.0 mg, or the quercetin standard of 2.0 mg, or the pseudohypericin standard of 1.0 mg, or the hypericin standard of 1.0 mg are dissolved to prepared stock solution, respectively. According to the guide of International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use , the sigp < 0.05 were considered as statistically significant, and p < 0.01 were considered as highly significant.The number of the experiment repetition in all the experiment was three times. The experimental data was analyzed with SPSS 17.0 software . The results are expressed as the means ± standard deviation (SD). Differences between groups were evaluated using the one-way analysis of variance (ANOVA) of two tailed test. The cell viability after trypan blue staining was observed under optical microscope and further confirmed by MTT method. The results showed that the maximum nontoxic concentration of HPE, HPW, and SEE was 78.13 μg/ml. When the concentration of HPE, HPW, and SEE was lower than 78.13 μg/ml, trypan blue staining showed cell was survival Figure 1B−5.8 TCID50 per 100 μl at 36 h post-inoculation  appeared at 36 h after IBV infection Figure 2Aculation Figure 2C50 to analyze the antiviral effect of HPE, HPW and SEE . Compared with the IBV group, the pathological scores of trachea and kidney in the HPE-TH group and the HPE-TM group decreased highly significantly (p < 0.01), and the score was similar to that of the RT group. At the same time, with the increase of HPE dose, the score decreased in a dose-dependent manner. This indicated that HPE had better anti-IBV effect.As can be seen from To confirm the inhibitory effect of HPE, IBV mRNA levels in trachea and kidney of different groups were measured by real-time qRT-PCR. As shown in via the MDA5 signaling pathway.It is well known that type I interferon, including IFN-α and IFN-β, plays a very important role in antiviral activity. As can be seen from At the same time, it is well known that inflammatory factors play an important role in inflammation. It also plays a very important role in IBV infection. As can be seen from + and (M-H)−]  (M-H)−] Figure 12 (M-H)−]  and foun2R = 0.9993–0.9997). The LOD (S/N = 3) of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin was in the range of 0.64 to 0.78 μg/ml. The LOQ (S/N = 10) of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin was in the range of 0.16 to 0.22 μg/ml. By measuring the peak area and retention time of standard compounds to evaluate the intra-day and inter-day precision, their relative SD was less than 1%. The results showed that the HPE contained 0.92% hyperoside, 1.37% quercitrin, 10.16% quercetin, 2.55% pseudohypericin, and 3.46% hypericin.The calibration curves of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin were as followed: y = 20,579x + 25,971, y = 18,877x + 41,654, y = 250,933x−272,745, y = 76,343x + 16,472, and y = 73,815x + 3,490.7, where y is peak area, x is sample concentration, and the concentration ranges were 10–60, 40–140, 10–60, 1–6, and 6–36 μg/ml, respectively. And the calibration curve was in accordance with a linear regression . qRT-PCR is a method of measuring the total amount of products after each reaction cycle with fluorescent chemicals in DNA amplification. IFA is a method to detect the presence of specific antigens in cells after the cells were treated with fluorescent-antibodies. TCID50 is the amount of infection in which 50% of the cells are infected by the virus. The anti-virus effect of drugs is often detected by qRT-PCR -like receptors (RLRs) in pattern recognition receptors (PRRs), it is well known that MDA5 plays a crucial role in avian respiratory disease progression (Some studies have shown that  and HIV , suggestgression , whose fgression , activatgression , leadinggression . NF-κB sgression . It is wgression .H. perforatum extract significantly downregulated the concentration of IL-6 and TNF-α in lung tissue for mice infected with an influenza A virus (in vitro and in vivo (Several reports have pointed out the MDA5 signaling pathways and innate immune cytokines were activated after infection with IBV M41 strain . MDA5 si A virus . In this in vivo 10. Mean− (−. This corresponds to the fragmentation regularity of quercitrin (− at 301.72 m/z confirms the presence of the quercetin (+ (− at 519.08 m/z (− at 505.44 m/z (− at 503.33 m/z (Because of remarkable antiviral effect on IBV, we analyzed the active components of HPE. The mass spectrum of hyperoside contained a negative quasi-molecular ion at 463.74 m/z [M–H]− . The masercitrin . Moreoveuercetin  (Figure H. perforatum, containing hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, possess anti-IBV activities. Furthermore, its anti-IBV effect may be associated with reduced mRNA expression levels of the pro-inflammatory cytokines IL-6, TNF-α by NF-κB signaling pathway, and related to up-regulate mRNA expression levels of type I interferon through the MDA5 signaling pathway, and could be useful for the development of new antiviral agents. However, further studies are required to elucidate its detail mechanism of action.Our study provides, for the first time, clear evidence that the extract of All datasets generated for this study are included in the article/in vitro experiments. RZ, XH, LD, HL, XL, XS, and GA participated in the animal experiments. GL designed the experiment and made valuable revision. All authors have read and approved the final version.HC wrote the manuscript and carried out most of the studies. IM, YZ, and YR participated in the This research was supported by the National Science and Technology Supporting Projects 31172295 and 31272569 operated by the Ministry of Science and Technology of China. In addition, this project belonged to Key Subject of Traditional Chinese Medicine of Jilin Agricultural Science and Technology University.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.


Ice-binding proteins (IBPs) are found in many organisms, such as fish and hexapods, plants, and bacteria that need to cope with low temperatures. Ice nucleation and thermal hysteresis are two attributes of IBPs. While ice nucleation is promoted by large proteins, known as ice nucleating proteins, the smaller IBPs, referred to as antifreeze proteins (AFPs), inhibit the growth of ice crystals by up to several degrees below the melting point, resulting in a thermal hysteresis (TH) gap between melting and ice growth. Recently, we showed that the nucleation capacity of two types of IBPs corresponds to their size, in agreement with classical nucleation theory. Here, we expand this finding to additional IBPs that we isolated from snow fleas (the arthropod Collembola), collected in northern Israel. Chemical analyses using circular dichroism and Fourier-transform infrared spectroscopy data suggest that these IBPs have a similar structure to a previously reported snow flea antifreeze protein. Further experiments reveal that the ice-shell purified proteins have hyperactive antifreeze properties, as determined by nanoliter osmometry, and also exhibit low ice-nucleation activity in accordance with their size. Collembola  are the world’s most abundant hexapods (arthropods and insects) and can be found in diverse habitats, from the tropics to the poles . CollembOnychiurus arcticus [Belgica antarctica [Studies on Collembola from Israel have been conducted for almost 100 years , mostly arcticus ,10 and ttarctica ,12.In the current study, we identified the species of the snow flea we found in the Hermon mountain, compared their protein characteristics to known snow flea proteins ,7, and cSnow fleas were collected from a valley on Mount Hermon, Israel  at 1450 m above sea level in January 2019 using insect aspirators. Approximately 2000 specimens of Collembola (a mixture of adults and juveniles) were kept in a few glass jars  was used to extract genomic DNA from mitochondria-rich tissues. The 710 bp region of the mitochondrial genome (cytochrome oxidase I (COI)) barcoding region was amplified using a polymerase chain reaction (PCR). The primer sequences used were:“LOC1490”5′-ATTCAACCAATCATAAAGATATTGG-3′ and “HCO2198”5′-TAAACTTCTGGATGTCCAAAAAAC-3′ . Briefly, the reactions were carried out in 10 μL volumes in a polymerase chain reaction (PCR) thermal cycler  equipped with a 96-well under the following conditions: A first cycle of 95 °C for 5 min followed by 5 cycles of 94 °C for 30 s, annealing at 45 °C for 30 s and 72 °C for 1 min, then 39 cycles of 94 °C for 30 s, annealing at 60 °C for 30 s, and, finally, 72 °C for 5 min. The PCR products were viewed with 1.2% agarose , The morphological analysis of the springtail samples were carried out using a Cryo-SEM device  at a high voltage of 5 kV. All the samples were rapidly frozen to reduce ice-crystal damage and improve the preservation of the specimens. The specimens were subsequently sputter-coated with carbon to avoid any charging effects. The snow fleas were homogenized in 50 mM Tris-HCl, 150 mM NaCl pH 7, and 1 mM phenylthiocarbamide, containing phenylmethylsulfonyl fluoride (PSMF), and subsequently centrifuged to separate the protein fraction from the extracellular matrices. After centrifugation, the supernatant was added to 50 mM Tris-HCl pH 8 buffer. A 100 kDa molecular weight cut-off centrifugal filter  was used to separate molecules of distinct sizes in the crude snow flea extract . A stockThe snow flea AFP was purified using the ice-shell affinity purification method . We usedSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted in a 6% stacking gel and a 16% solving gel following a previously-used procedure . Mass sp−1 to 1600 cm−1 with a spectral resolution of 1 cm. All the samples were air-dried for 30 min inside a laminar flow bench before the FTIR analysis.Fourier-transform infrared (FTIR) spectroscopy of the samples was undertaken using an FTIR spectrometer  in total reflection mode. The experiment was performed as previously discussed in the literature ,22. SpecThe >100 kDa fraction and the <100 kDa that was further purified on ice-shell fraction (that contained the ice-binding proteins (IBPs)) were dialyzed against 20 mM sodium phosphate at pH 7. Circular dichroism (CD) spectroscopy  was performed at 4 °C. The spectrum was analyzed using DichroWeb ,24.We used a custom-designed nanoliter osmometer system with a temperature controller  and a customized cooling stage, previously described by . The tem−1 and the freezing temperature was determined automatically using an image analysis LabVIEW program for each individual droplet based on the sudden decrease in the droplet’s brightness during freezing.Ice nucleation measurements were performed using the Weizmann Supercooled Droplets Observation on a Microarray (WISDOM) setup, detailed by ,25,26. A50, for pure water droplets, T50(wat); pure buffer droplets, T50(buf), and droplets containing various concentrations of IBP in the buffer, T50(IBP). The Tris buffer had a significant effect on the T50 temperatures (T50(wat) = −35.7 ± 0.4 °C and T50(buf) = −38.2 ± 0.4 °C) because the solutes decreased both the ice melting point and the homogeneous ice nucleation temperature [het(IBP) with previous measurements, as well as with theoretical predictions of the critical ice embryo size in pure water, the measured ice nucleation temperatures were corrected for the effect of the buffer, similar to previous studies [The experimental ice nucleation data were analyzed as described previously . From thperature ,27. Ther studies  as follohet(IBP) is slightly shifted, similar to the effect by which the buffer decreases the T50(wat) of pure water. Note that this treatment also accounts for the effect on freezing temperature, with different buffers or different buffer concentrations. It also allows for a comparison of the heterogeneous ice nucleation temperature and the corresponding critical ice embryo size determined from classical nucleation theory, which is usually calculated for pure water and not for buffer solution. Moreover, a comparison between previously measured ice nucleation temperatures of IBPs and the critical ice embryo from classical nucleation theory (CNT) requires an estimate of the three-dimensional size of the IBP, which was previously estimated from its molecular weight and the protein density, assuming a cubic protein shape, see Eickhoff et al. (2019) for details and corresponding parameterizations [Hence, the Tizations . For theizations ,29,30, s(AT) bias , and the Guanine and Cytosine (GC) content was 37.3%. An AT nucleotide bias frequently found in hexapods was also identified. The percentage of AT (62.7%) found in the current study was close to the percentage of 63–64% reported for Antarctic Collembola [The mitochondrial genome COI 676-bp fragment has been sequenced  tree, featuring the captured Collembola COI sequence, along with reference COI genes from the NCBI database, showed five major clusters corresponding to the Protaphorura, Brachystomellidae, Hypogastrura, Homidia, and Entomobrya families. There were no anomalies among any of these classes, and all the recognized reference samples clustered properly . The seqThe specific feature of the Collembolan’s cuticle is the comb-like structures described by Helbig et. al. . These sIn the current study, we found that the collected springtails possessed secondary granules, typical of the cuticle structure of the order Poduromorpha. These secondary granules, exhibiting a basic hexagonal structure, are seen as an adaptation to living in soil ,34. The Based on the centrifugation and size exclusion, the proteins in the snow fleas crude extract were separated. The extract was divided into two fractions: <100 kDa and >100 kDa  and chemical (FTIR and CD spectroscopy) characterizations.−1 and 1520–1590 cm−1, which are characteristic bands for the amide I and amide II vibrations, respectively [−1 that are characteristic of saccharide moieties, such as cellulose [−1, which indicated the presence of an alpha helix and an unordered structure, respectively [−1. The peak at 1654 cm−1 confirmed the presence of an alpha helix. The existence of the amide I  and amide II  bands at wavenumber intervals clearly showed that there was a significant amount of secondary structure-oriented components in the IBP. An absorption band between 1030 and 1040 cm−1 was observed in all the samples due to traces of alcohol remaining after the cleaning of the sapphire slide. The FTIR results for the large fraction are shown in The ice-affinity purified fraction was further investigated using FTIR spectroscopy in total reflection mode . The samectively . The samellulose , which iectively . The IBPThe secondary structure of the protein fractions was determined using CD spectroscopy at 4 °C. In-depth secondary structure analysis was carried out using the Dichro Web Server to plot the CD spectra. The IBP fraction exhibited a typical spectrum, with minima at 195 nm and maxima at 210 nm see . A similAs shown in 50, where 50% of the droplets of pure 50 mM of Tris-HCl and 150 mM of NaCl pH 7 buffer froze and was determined to be −38.2 ± 0.4 °C, which, due to the colligative effect of the dissolved salts, was colder than the T50 (−35.7 ± 0.4 °C) of pure water droplets.The ability to initiate ice in supercooled nanoliter droplets was determined using the WISDOM setup for IBP solutions of various concentrations in a Tris buffer. The median freezing temperature, Tice, as a function of the temperature of the IBP fraction. Four concentrations of IBP were investigated, ranging from 0.03 to 0.5 mg mL−1, and a clear dependence of the freezing temperature on the IBP concentration was observed. Higher concentrations exhibited higher freezing temperatures with T50 values ranging from −37.2 to −35.7 °C. In m) per IBP mass as a function of temperature. Higher nm values reflect a more active ice-nucleating material. The nm is based on the cumulative fice function and was calculated considering the mass of IBP per droplet to account for the effect of IBP concentration on the freezing activity [ice in m curve ; both were recently measured also using WISDOM [TmAFP, respectively) to the main IBP extracted from the Collembola and also observed on the SDS-PAGE gel (6.5 kDa), which suggests that their active sites for ice nucleation are of a similar size, estimated to be about 2 nm. In detail, we estimated the size of the ice-binding site of the 6.5 kDa Collembola IBP using the structure given by [1 = 1.7 nm and d2 = 4.7 nm. In order to represent these two ellipsoidal diameters by a single equivalent spherical diameter deq that can be compared to the spherical ice cap calculation from CNT, we use a version of the Young–Laplace equation, eq = 2.5 nm. Hence, we estimate the size of the 6.5 kDa IBP binding site to be between 1.7 and 2.5 nm, and we have indicated that range in As presented in g WISDOM . Both ofPAGE gel .5 kDa, wgiven by . The recCollembola species are ubiquitous and their location may play a significant role in their structure and genetics . In thisIn order to confirm the morphological characteristics from a taxonomic aspect, phylogenetic analysis was carried out using the COI gene. The COI gene is the most targeted mitochondrial DNA gene due to its high intraspecific diversity, which give it a well-established molecular identification . The morVarious studies of Collembola (and other hexapods) that live in cold climates have indicated the presence of ice-nucleating agents and hyperactive AFPs . Here, a−1 region. The ice shell purified fraction showed spectra at 1600–1700 cm−1 and 1520–1590 cm−1, which indicated that the ice-binding portion exhibits bands specific to an alpha helix. This result is in accordance with the previously reported ice-binding protein extracted from snow fleas [FTIR spectroscopy analysis ,36 was uow fleas . We alsoWe examined the TH activity, an important characteristic of AFPs, of our samples. The experiments reveal that the IBPs have a high TH activity of more than 3 °C at a concentration of 1 mg/mL, which confirms that the ice-shell purified proteins are hyperactive AFPs. Our study shows that the crystal shape obtained in the presence of IBPs during melting is consistent with our previous studies on hyperactive antifreeze proteins .1 = 3.4 nm and d2 = 4.6 nm, resulting in an equivalent spherical diameter of deq = 3.9 nm. We have added this size range as the cyan vertical range in In this study, we have shown for the first time the ice nucleation activity of snow flea IBPs. The INA increased with the concentration of IBP in the solution, which led to warmer nucleation temperatures and a higher number of nucleation events at a particular temperature. This study supports previous findings of the contrasting behaviors of AFPs that can inhibit the growth of existing ice crystals and can also initiate the nucleation of new ice crystals in supercooled solutions ,42,48. AA new Collembola species was found in Mount Hermon, a snow flea which contains ice-binding proteins (IBPs). The SDS-PAGE and mass spectrometry revealed the presence of a 6.5 kDa IBP, and mass spectrometry also showed small amounts of a 15.7 kDa IBP. These protein masses, as well as the CD spectrum, are similar to those of IBPs extracted from snow fleas collected in Canada [


Influenza B viruses (IBVs) are major contributors to total human influenza disease, responsible for ~1/3 of all infections. These viruses, however, are relatively less studied than the related influenza A viruses (IAVs). While it has historically been assumed that the viral biology and mechanisms of pathogenesis for all influenza viruses were highly similar, studies have shown that IBVs possess unique characteristics. Relative to IAV, IBV encodes distinct viral proteins, displays a different mutational rate, has unique patterns of tropism, and elicits different immune responses. More work is therefore required to define the mechanisms of IBV pathogenesis. One valuable approach to characterize mechanisms of microbial disease is the use of genetically modified pathogens that harbor exogenous reporter genes. Over the last few years, IBV reporter viruses have been developed and used to provide new insights into the host response to infection, viral spread, and the testing of antiviral therapeutics. In this review, we will highlight the history and study of IBVs with particular emphasis on the use of genetically modified viruses and discuss some remaining gaps in knowledge that can be addressed using reporter expressing IBVs. Influenza B viruses (IBVs) are negative-sense, segmented RNA viruses of the Orthomyxoviridae family . IBV wasIBV infection causes acute respiratory disease including the induction of the innate immune response which is associated with fever, body aches, and fatigue; a set of symptoms collectively termed “influenza-like illness” ,6. PrimaThe historical lack of focus on IBVs is likely due to an incomplete understanding of the prevalence and disease severity of IBV. Until recently, influenza subtypes isolated from human clinical infections were differentiated on the basis of hemagglutination inhibition testing, a process that lacks sensitivity . It wasnNB and BM2  structures and differential affinities for host glycans ,34. DiffOur current understanding of IBV biology and mechanisms of pathogenesis, though incomplete, are derived from a number of different scientific approaches. Previous studies have used “mini-genome” replicase assays to study aspects of the viral life cycle related to replication ,46. The Currently, we can make use of viral reverse genetic approaches to generate viral tools that allow us to define how influenza viruses interact with their host and cause disease. Multiple fundamental discoveries, however, were crucial in enabling the development and use of these systems ,54. Due In 1999, the first reports of reverse genetics systems for IAVs were published ,59. UsinNB, BM2 genes expressed by IBV. Starting in 2003, Hatta et al. reported that the influenza NB protein, an accessory protein encoded on segment 6 that is not conserved in IAVs, could be knocked out and therefore shown to be unnecessary for B/Lee/40 viral replication in vitro [Since the development of the IBV reverse genetic system, several groups have used genetically modified viruses to study IBV biology, highlighting the unique features such as the in vitro . Furtherin vitro .It was also during the years following the introduction of reverse genetics for IBV that the function for the IBV BM2 protein, which is encoded on segment 7, was discovered. In 2004, several groups used reverse genetic systems to demonstrate that BM2 was necessary for incorporation of viral ribonucleoprotein complexes into virions and replication ,65. AddiMutant viruses have also been used to probe the mechanisms of innate immune evasion of IBVs. In the B/Lee/40 viral background, it was discovered that the nonstructural protein (NS1) was essential for viral growth. These studies demonstrated that, despite the lack of protein homology between IAV NS1 and IBV NS1, the interferon antagonizing functions are conserved ,70. LateAlthough the IBV genome could be genetically modified as early as the late 1990s, there was a lack of viral strains that harbored reporter proteins for rapid and sensitive detection of viral infection. Hatta et al. were the first group to insert reporter genes into the IBV genome, albeit at the expense of the virus losing full replication potential. In this work, the authors found that the IBV neuraminidase (NA) open-reading frame (ORF) could be replaced by green fluorescent protein (GFP) and viral spread could occur if the requirement for neuraminidase enzymatic activity was satisfied via addition of exogenous sialidases .In order to develop fully infectious reporter IBV strains, knowledge from the development of reporter IAVs ,79,80,81Shortly after this report, Breen et al., designed and rescued a replication-competent reporter IBV which contained a Timer fluorescent reporter which undergoes a spectral shift over the course of time . In the Most recently, Dumm et al. published a paper using the B/Malaysia/2506/2004 background to express Cre recombinase in the PB1 segment of the genome h. The auThe viruses described in these reports all have advantages and limitations based on their specific design and the reporter protein expressed (described in The recombinant viral tools generated in the last 20 years have facilitated many important discoveries, however important scientific questions remain. Some of the most pressing concerns in the influenza field are the design of effective vaccines to prevent disease and the discovery of additional antiviral therapeutics. While modification of the virus itself to function as a better vaccine has been reviewed elsewhere ,54,61, rThere are also basic science questions which remain to be answered. IBV generally presents as an infection of the upper respiratory tract. However, a feature which results in severe influenza disease is lower respiratory tract involvement leading to acute respiratory distress syndrome . Much ofFinally, there are fundamental aspects of viral biology, ranging from the functions of viral proteins to the mechanisms controlling viral assembly and reassortment that remain incompletely characterized for influenza viruses in general . ResearcIn conclusion, genetically modified and reporter viruses add to the molecular virologists’ toolbox and have already been used to answer important questions about IBV biology. More work is necessary, however, to expand the available virus repertoire and engineer novel viruses to increase our growing understanding of IBV and facilitate the development of next generation vaccines and antiviral therapeutics.