These events have become more frequent during the twenty-first century, with 5–10% of total northeast Greenland melt in several recent summers occurring during the ~1% of times with strong AR and foehn conditions. We conclude that the combined AR-foehn influence on northeast Greenland extreme melt will likely continue to grow as regional atmospheric moisture content increases with climate warming.The Greenland Ice Sheet has been losing mass at an increased rate in recent decades. In northeast Greenland, increasing surface melt has accompanied speed-ups in the outlet glaciers of the Northeast Greenland Ice Stream, which contain over one meter of sea level rise potential. Here we show that the most intense northeast Greenland melt events are driven by atmospheric rivers (ARs) affecting northwest Greenland that induce foehn winds in the northeast. Near low-elevation outlet glaciers, 80–100% of extreme (> 99 Extreme ice sheet melt events in northeast Greenland occur after intense water vapor transport into northwest Greenland by atmospheric rivers. Through the foehn effect, the air becomes warmer and drier as it descends the ice sheet slope. In the last decade, the largest melt anomalies and overall mass loss acceleration have shifted poleward from southern to northern Greenland3. After the unprecedented ice sheet-wide melt episode in July 2012, a number of recent major melt events have disproportionately affected northern Greenland6.The Greenland Ice Sheet (GrIS) has lost ~3.9 billion tons of ice since 1992 and contributed 10.8 mm to global mean sea level rise, of which more than 50% is attributed to surface mass loss due to increased meltwater runoff7. NEGIS outlet glaciers have exhibited increasing mass loss in recent years, due to warming air and ocean temperatures leading to the loss of buttressing sea ice and ice shelf collapses at the floating glacier margins13.East of the northern Greenland orographic divide, solid ice flow is dominated by the fast-flowing Northeast Greenland Ice Stream (NEGIS). The NEGIS drains ~16% of the GrIS and terminates at three primary marine outlet glaciers  that collectively contain over 1 m of potential sea level rise14 that forms in NE Greenland during the summer. Ice sheet surface melt events have been linked to summer speedups of 79°N Glacier, and glacier acceleration after supraglacial lake drainage at the end of the melt season has also been observed16. Extreme melt events decrease the ice sheet albedo and precondition the surface for enhanced subsequent melt18. Surface runoff may also be influenced by the development of near-surface ice slabs which may persist for years after short-lived extreme melt events and are found extensively in the NEGIS catchment19. These characteristics indicate that both the liquid runoff and solid ice flow components of NE Greenland’s sea level rise contribution are influenced by warming-induced surface melt, and particularly by extreme melt events with compounding short- and long-term impacts.Ice flow dynamics in northeast (NE) Greenland are linked to surface hydrology through the extensive network of supraglacial streams and lakes22. Mattingly et al.21 found evidence that atmospheric rivers (ARs) affecting northwest (NW) Greenland may lead to foehn conditions and enhanced melt in NE Greenland after the moist air mass crosses the ice divide and flows downslope, similar to warming documented on the lee side of the West Antarctic peninsula and other mountainous areas26. However, there has not been any systematic study of the role of foehn in forcing melt in northern Greenland, where the topography consists of a wide dome with a relatively gentle slope in comparison with the more narrow mountain ranges studied in other regions.Several studies have suggested a link between intense NE Greenland melt events and warm, dry downslope winds known as “foehn” descending from the GrIS plateau to the westIn this study, we utilize AR detection algorithms, a foehn identification procedure applied to output from two regional climate models, and a high-resolution atmospheric model simulation to demonstrate that foehn winds induced by ARs drive the majority of extreme melt events in NE Greenland. We first describe the substantial contribution of NW Greenland AR events to NE Greenland melting, then analyze the relationship between ARs and foehn-induced extreme melt events, and finally quantify the contribution of this AR-foehn-extreme melt mechanism to the observed increasing trend in NE Greenland summer melt.The capability of ARs impinging on western Greenland to trigger melt in NW Greenland is exemplified by an intense AR landfall in NW Greenland on 20 July 2014 which triggered a downsloping foehn wind that produced significant melt in the NE region of the GrIS Fig. . The oro27 AR detection algorithm). Over the 48 h following an AR, the amount of melting associated with AR events increases at lower elevations to ~40–50% of total JJA melting : up to 75% of extreme melt events at low-lying elevations are associated with AR landfalls  when melt magnitude becomes negligible. A similar phenomenon is observed over the Antarctic Peninsula where a greater IVT upon landfall leads to a greater latent heat release, as the moisture is orographically lifted over the coastal mountain ranges and creates a more intense leeward foehn wind compared to non-AR related foehn events31.The rate of melting in NE Greenland increases as a function of AR intensity, where intensity is measured by the maximum integrated water vapor transport (IVT) upon landfall Fig. . ARs wit32  case study simulation of the NEGIS region using the Polar Weather Research and Forecasting (Polar WRF) model32 Fig. . Typical33 and Alpine valleys34. Alternatively, this could be a real effect related to lee waves associated with the foehn effect drawing cool air from the east, which could be investigated with more detailed simulations in future studies.Figure Having demonstrated the role of foehn winds in forcing NE Greenland melt during one example of a strong summer AR event in NW Greenland, we now examine the prevalence of foehn-induced melt and its association with ARs during the full climatological record (1980–2020). Significant surface melt in NE Greenland is primarily confined to the narrow, low-elevation (<1000 m) ablation zone along the ice sheet margins Fig. . The exc90 events), the majority of melt (50–65%) in the middle and lower NEGIS catchment occurs during foehn  NW Greenland AR landfalls   events in NW Greenland during 1980–2020  with the NAO index. Melt occurring with foehn and no AR90 is the second most common category (14.97%) and is characterized by a step function-like increase around 2000, with several JJA seasons after 2000 having a large percentage of melt attributed to foehn  can generate foehn-induced melt in NE Greenland.The most frequent melt condition is “no ARime Fig. . This coehn Fig. . Foehn c90 events  calculated from 6-h Modern-Era Retrospective analysis for Research and Applications, Version 2 (MERRA-2) reanalysis data, which has 0.5° × 0.625° horizontal resolution47. IVT (in units of kg m−1 s−1) is calculated using the formulag (m s−2) is the gravitational acceleration, q (kg kg−1), and V (m s−1) are the specific humidity and vector wind, respectively, at a given atmospheric pressure level, and dp is the increment between pressure levels . The algorithm first detects areas where IVT is greater than a minimum threshold of 150 kg m−1 s−1 and exceeds the 85th climatological percentile, where climatology is defined by the distribution of all values during 1980–2016 within a 31-day centered window at a given grid point. Areas where these conditions are met are then filtered by several size, shape, and location criteria to ensure the final AR record is composed of long, narrow moisture transport features in the Northern Hemisphere mid-latitude and polar regions. The algorithm also requires moisture transport to be in the poleward direction for mid-latitude ARs, but relaxes this requirement for ARs north of 70°N in order to detect moisture plumes originating in the Arctic that may affect Greenland. We tested a version of this detection algorithm forced with ERA5 reanalysis and found a negligible difference in AR detection frequency and AR-related melting. Further details on this AR detection method can be found in refs. 27.Two different AR detection algorithms are employed in this study for comparison purposes. The first algorithm was developed by ref. 31. This algorithm identifies grid cells between 42.5° and 85°N for values of vIVT  component of IVT) within the 98th percentile of all monthly vIVT values from 1980 to 2020. If a filament of vIVT values within this percentile extends at least 20° in the meridional direction, it is identified as an AR. The vIVT (kg m−1 s−1) term was calculated asq, g, and dp are the same as described above, and v is the meridional component of wind velocity (m s−1). Like other AR detection algorithms in the Atmospheric River Tracking Method Intercomparison Project (ARTMIP), this algorithm is applied to 3-hourly fields on all reanalysis levels from MERRA-2. For additional details regarding this AR detection algorithm, see ref. 31.The other AR detection method utilized in this study is a polar-specialized algorithm originally developed for use in the Antarctic by refs. 48. Here RACMO2 is run at 5.5 km spatial resolution over a domain including the GrIS, its peripheral ice caps, and glaciers of the northern Canadian Arctic, Svalbard and Iceland3. The model incorporates the dynamical core of the High Resolution Limited Area Model (HIRLAM) and the physics package of the European Centre for Medium-Range Weather Forecasts-Integrated Forecast System (ECMWF-IFS). RACMO2 includes a 40-layer snow module (up to 100 m depth) simulating melt, percolation and retention into firn, and runoff. It represents dry-snow densification, drifting snow erosion, and snow albedo based on grain size, cloud optical thickness, solar zenith angle, and impurity content (soot).RACMO2 is specifically adapted to represent surface processes in polar regions48.RACMO2 is forced by a combination of climate reanalyses including ERA-40 (1958–1979), ERA-Interim (1980–1989), and ERA5 (1990–2021) within a 24-grid-cell-wide relaxation zone at the model lateral boundaries. Upper atmospheric relaxation is also active. Forcing consists of temperature, pressure, specific humidity, wind speed and direction being prescribed at the 40 model atmospheric levels every 6 h (ERA-40 and ERA-Interim) or 3 h (ERA5). Sea ice extent and sea surface temperature are also prescribed from the ERA reanalyses on a 6-hourly or 3-hourly basis. Firn is initialized in September 1957 using snow temperature and density profiles from the offline Institute for Marine and Atmospheric research Utrecht-Firn Densification Model (IMAU-FDM). Ice albedo is prescribed from the 500 m Moderate Resolution Imaging Spectroradiometer (MODIS) 16-day surface albedo product (MCD43A3) as the lowest 5% annual values averaged for the period 2000–2015, ranging from 0.30 for dark bare ice to 0.55 for clean bright ice under perennial firn. For detailed model description and evaluation using in situ and remote sensing measurements, we refer to refs. 49. The version 3.12.0 of MAR has been run here at a resolution of 15 km, 1-hourly forced by the ERA5 reanalysis since 1950. With respect to MARv3.11 (fully described in ref. 50), the main improvements of MARv3.12 are the geographical projection used by MAR which is now the standard Polar Stereographic EPSG 3413, a correction of an important bug impacting the snow temperature at the base of the snowpack, a conservation of water mass in the soil impacting water fluxes over the tundra, and a continuous conversion from rainfall to snowfall from 0 °C to −2 °C as input of the snow model instead as a fixed one at −1 °C.MAR is a regional climate model especially developed for studying the near surface climate and SMB of both polar ice sheets where MAR has been widely validated with in situ observations and satellite data sets. With RACMO2, it is notably one of the best models currently available for simulating the GrIS SMBMAR ice mask is a percentage value, so the foehn algorithm is only applied where permanent ice covers >10% of a grid cell. In RACMO2, the ice mask is binary, with SMB components only calculated for grid cells with ice coverage ≥50%. We use the version of the RACMO2 mask which includes peripheral ice caps, so that we can include Flade Issblink in our analysis.51 during 1980–2020. This dataset classifies the ice sheet surface into melt/no melt areas with daily temporal resolution, based on brightness temperature data acquired by three passive microwave radiometers: the Scanning Multichannel Microwave Radiometer (SMMR), the Special Sensor Microwave/Imager (SSM/I), and the Special Sensor Microwave Imager/Sounder (SSMIS). This dataset has in turn been compared to Greenland station observations of melt conditions and compares favorably to other melt retrieval algorithms applied to passive microwave satellite data52.To evaluate the RACMO2 and MAR melt simulations, we used the NASA MEaSUREs Greenland Surface Melt Daily 25 km EASE-Grid 2.0 dataset53 or 8.25 mmWE/day55 to classify “melt days” using MAR, so we tested the following meltwater production thresholds: 0.1, 1, 2, 5, 8.25, 10 mmWE/day.In order to compare the model and satellite data, we first bilinearly interpolated the RACMO2 and MAR melt data to the 25 km EASE Grid 2.0 on which the MEaSUREs melt data is provided, and masked out grid cells in the NE Greenland domain that are not classified as ice sheet by all three datasets. Since the passive microwave melt data is provided as binary melt/no melt values with daily temporal resolution, whereas RACMO2 (MAR) outputs meltwater production values with 3-hourly (hourly) resolution, we developed a method to compare the model meltwater production values with the binary satellite values. We summed the RACMO2 & MAR meltwater production values for each day, then tested a range of threshold values of meltwater production (mmWE/day) for classification as a “melt day” in the model datasets. Previous studies have used a threshold of 1 mmWE/dayAccuracy: (TP + TN)/totalTrue positive rate: TP/actual yesFalse positive rate: FP/actual noTrue negative rate: TN/actual noUsing all available values from ice sheet grid cells in the NE Greenland domain during JJA 1980–2020, we produced a confusion matrix for each model melt threshold. The confusion matrix classifies each pixel on each day into one of four categories: “true negative” ; “false positive” ; “false negative” ; and “true positive” . Then we calculated the following summary statistics from the confusion matrix at each threshold:The validation summary statistics are provided in Supplementary Table Finally, to evaluate how well RACMO2 and MAR simulate the spatial pattern of NE Greenland melt, we created maps of the overall percentage of JJA days with melt for each dataset and their differences with the MEaSUREs data. The plots for the 1, 2, and 5 mmWE/day thresholds are shown in Supplementary Figs. 56. Assumptions are made that foehn has a sufficiently distinct signature at a given location to be identified using (1) thresholds of key meteorological fields , (2) changes in conditions from pre- and post-foehn conditions  or (3) a combination of both  to capture the foehn onset and development, as well as their features throughout the event. No Foehn-Identification Algorithm (FIA) previously existed for Greenland, therefore the following method is developed for detection of foehn in MAR and RACMO2.The classification of foehn events is non-trivial, and there is no established best practiceWind direction between 220° and 350° AND−1 ANDWind speed greater than 5 m sA relative humidity value less than the 15th percentile of a 2-week window surrounding the given date ORA 5% decrease in relative humidity AND 3 °C increase in temperature compared to the previous 6-h valueFor grid cells where the ice mask threshold is greater than 10%, foehn is detected from six-hourly averaged values when the following conditions are simulated:57). The 15th percentile for relative humidity is chosen based on our exploratory case study analysis of foehn events in both AWS data from KPC_L and KPC_U, and MAR data (not shown). This threshold detects most foehn cases whilst excluding other westerly flows such as katabatic winds. Note that this FIA is developed specifically for NE Greenland and would require modification for other areas of Greenland where downslope winds are not westerly in direction.The westerly wind requirement removes the possibility of including warm air flow from the Atlantic with passing storms. The combination of relative humidity decrease and simultaneous temperature increase reduces the possibility of including cold katabatic winds in the results, and follows similar FIAs in the Antarctic  model was previously simulated and evaluated by ref. 90 and foehn conditions are fit with trend lines using the nonlinear Ensemble Empirical Mode Decomposition (EEMD) method (ref. 58) using the Rlibeemd package for the R programming language59. Correlations between anomalies in NAO/GBI and melt values are calculated by subtracting the trend fit from each time series. We present only the NAO results in the paper, as the melt time series correlations with GBI are qualitatively similar but with a reversed sign, and we also note that correlations are similar using the actual (non-detrended) values of each variable.The time series of NAO, Greenland Blocking Index (GBI), and melt attributed to the four possible combinations of ARSupplementary InformationPeer Review FileSupplementary Movie 1


Infectious bronchitis virus (IBV) is an enveloped and positive-sense single-stranded RNA virus. IBV was the first coronavirus to be discovered and predominantly causes respiratory disease in commercial poultry worldwide. This review summarizes several important aspects of IBV, including epidemiology, genetic diversity, antigenic diversity, and multiple system disease caused by IBV as well as vaccination and antiviral strategies. Understanding these areas will provide insight into the mechanism of pathogenicity and immunoprotection of IBV and may improve prevention and control strategies for the disease. Coronaviridae within the order Nidovirales and are widespread pathogens of significant potential threat to animals and humans [Coronaviridae are divided into alpha-, beta-, delta-, and gammacoronavirus genera (Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses belonging to the family d humans ,2,3. Thes genera  and can Among these CoVs, the porcine epidemic diarrhea virus and transmissible gastroenteritis virus of the Alphacoronavirus genus can impose a heavy disease burden on swine. Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, which belong to the Betacoronavirus genus, have a significant mortality rate in humans. Porcine deltacoronavirus of the Deltacoronavirus genus infects piglets causing diarrhea, vomiting, and dehydration. Infectious bronchitis virus (IBV) of the Gammacoronavirus genus causes a highly contagious respiratory, renal, and genital disease in chickens known as avian infectious bronchitis (IB) ,2,5,6.IB is now a predominantly viral disease of commercial poultry worldwide that can affect chickens of all ages and types and has become a significant challenge for the poultry industry globally despite the extensive use of vaccination as a major control measure ,9,10,11.IBV was the first coronavirus to be discovered and was isolated from newly hatched chicks in the USA in 1931 . The natIn the USA, numerous IBV strains have been isolated since the 1930s, and major lineages belong to GI-9 (ArkDPI-like strains), GI-17 (DMV/1639-like strains), and GI-25 (CA1737-like strains) ,53. CurrEspecially in China, IBV was first described and isolated from chickens in the early 1980s. The virus has now spread to all poultry breeding areas. IB ranks first or second among the important infectious diseases of poultry in annual statistics in China and causes significant economic losses. Currently, at least four main IBV lineages, including QX-type (GI-19), Taiwan-I (GI-7), TC07-2 (GⅥ-1), and 793B-type (GI-13) coexist in chicken farms, which considerably increases the difficulty of prevention and control of the disease ,43,44,54High rates of mutation, short generation times, and large population sizes drive the rapid evolution of RNA viruses, and consequently, most of the viral outbreaks over the last 100 years have been caused by RNA viruses . Among tIBV strains are clustered into six genotypes (GⅠ–GⅥ) using the complete nucleotide sequences of the S1 gene, and together these contain 34 distinct viral lineages and several inter-lineage recombinants . Of thesA timeline with the date of isolation of each presenting IBV strain and used live-attenuated vaccines in China is shown in New IBV variants continuously emerge through genetic recombination and mutation, and several exhibit clear antigenicity differences revealed by cross-neutralization tests or monoclonal antibody analysis. The GI-19 lineage (QX-type) strains, for instance, are prevalent in many parts of the world, and are distinct from all other known IBV strains, indicating that these strains belong to different serotypes ,72,73. ACurrently, IBV exists as a serotype score worldwide, and most of these have poor cross-protection with each other ,75,76,77Most IBVs can infect the respiratory, renal, and reproductive systems of chickens. Following the initial infection in the respiratory tract with clinical signs , IBV is disseminated to other tissues by viremia . One posAlthough differences in clinical and pathological outcomes in chickens depend on the infecting IBV strain, many IBV strains can simultaneously infect multiple physiological systems in chickens, which is termed “multiple symptoms with one virus” . First, In modern poultry farms, an effective management system and biosecurity measures are of primary importance to control infectious diseases. This premise is based entirely on the correct understanding of the factors influencing viral spread, as with the avian influenza virus ,101,102.Despite drawbacks, including severe reactions to vaccination at the day of age, the likelihood of viral recombination, and frequent vaccine replacement caused by viral mutation, vaccination remains the most effective control measure for IB ,108,109.Currently, almost all commercial chicken flocks are vaccinated against IBV. The most effective IB vaccine is the live-attenuated vaccine, and H120 is one of the excellent vaccine strains widely used in the world. The vaccination protocol for IBV can vary depending on the vaccine used and the specific conditions of the poultry operation. In general, chicks are vaccinated at one day old or at hatchery, and then again several times throughout their life to maintain immunity. Booster vaccinations may be given at 7–10 days of age, at 3–4 weeks of age, and then every 5–6 weeks thereafter. Because of the many existing variants worldwide, the concept of protectotype has been increasingly accepted for controlling IB. One of the most commonly applied protectotype vaccination protocols against IB in the EU is the simultaneous or alternate use of Ma5 and 4/91 vaccine strains to provide protection against homologous or heterologous IBV strains . In ChinDifferent vaccine platforms have been designed to develop effective vaccines against IBV. However, new vaccine techniques are rarely used because of the relatively complex immune protection mechanism and are currently only in the laboratory research stage ,116,117.IB is a highly contagious viral disease caused by coronavirus IBV that produces severe economic losses in the poultry industry worldwide. Despite immunization and biosecurity measures, IB still frequently occurs in commercial chicken flocks because of the constant emergence of new IBV variants as well as poor cross-protection against them with the existing vaccines currently in use. Therefore, similarly to the threat of SARS-CoV-2 to human health, IB continuously threatens the healthy development of the poultry industry.As with other CoVs, IBV undergoes rapid evolution, producing a growing number of genotypes and serotypes due to high rates of mutation, viral recombination, and host selection pressure. IBVs have a high genetic and antigenic diversity, whereas our knowledge of the ecology and evolution of IBVs remains very limited.IBV infection can induce obvious respiratory and reproductive symptoms as well as increased mortality in chickens, especially when secondary infections of bacteria or viruses occur. “Multiple symptoms with one virus” caused by IBV affects chickens of various types and ages globally and most likely will continue to be a serious threat to the global poultry industry.To prevent and control IB, the most effective measure at present remains vaccination despite current challenges. Clarifying the pathogenesis of the virus and optimizing the vaccination strategy using specific local strains will help to address continuing concerns over the efficacy of existing IB vaccines.


It was found that the unsaturated fatty acid  content of the samples containing oleogel was significantly higher (p < 0.01) than that of the samples containing milk fat. However, butyric and caproic acids, which are aliphatic fatty acids, were found to be deficient in the samples to which oleogel was added instead of milk fat. As to the microscopic appearance, while water and oil particles were not homogeneously dispersed in the ice cream samples with oleogel, they were fully homogeneously dispersed in the milk fat-added ice cream samples. In addition, it was determined that panelists preferred the samples with added milk fat as fat source, salep, and PG as emulsifier. Among the samples with added oleogel as the oil source, they liked the sample added with oleogel as fat source, konjac gum, and no emulgator more.This study investigated the possible usage of beeswax oleogels instead of milk fat as a fat source in ice cream production and konjac gum as a stabilizer instead of salep. For this aim, 12 different ice cream samples were prepared using various fat and oil sources (milk fat and oleogel), stabilizers , and emulsifiers (monoglyceride (MG), Palsgaard (PG), and no emulsifier/emulsifier-free (NE)). It was determined that the overrun ratio of ice cream samples containing milk fat was higher than that of samples containing oleogel, and the viscosity of the ice cream mix containing Palsgaard and oleogel was greater than that of the mix with other treatments . While the first dripping time of the samples with PG and konjac gum was longer compared to the samples without emulsifier (NE) or monoglyceride (MG), the complete melting times of the samples were close to each other. Whereas the dissolution rate of the samples with salep was higher than that of the samples with konjac gum, the fat destabilization ratios of the samples with oleogel and konjac gum were lower than those of the samples with milk fat and salep. The fat destabilization ratio of samples containing PG as an emulsifier was statistically higher ( Ice cream is a complex food with several structural components. Ice cream is a frozen dairy product that contains fat, sugar, non-fat milk solids, flavors, emulsifiers, and stabilizers. Ice cream is a fat-rich, delicious dairy product that is enjoyed by many people all over the world. Since ice cream contains a high percentage of milk fat (9%), consumers are more likely to develop obesity and cardiovascular disease. The production of medium-fat, low-fat, and non-fat ice cream reduces the risk of obesity and cardiovascular disease due to a lower intake of saturated fats . Fat redSome manufacturers want to reduce milk fat and use vegetable oils in ice cream formulation; therefore, a significant portion of ice cream can be made with naturally saturated oils such as palm kernel oil, palm oil, or coconut oil, which are commonly used for this purpose . Trans-fSolid fat plays an important role in maintaining the structure of ice cream, and a small amount of crystalline fat is required to maintain the instability that allows the structure to develop; therefore, solid fat in ice cream cannot be replaced with liquid oil. However, because saturated fat is linked to an increased risk of heart disease, the WHO recommends that saturated fat intake be constrained to no more than 10% of daily energy intake . The useStabilizers are used in ice cream production to increase the viscosity of the mix, prevent the formation of ice crystals during processing and storage, and preserve its structure by slowing melting during the consumption stage. In Turkey, salep is commonly used as a stabilizer in the production of ice cream. The use of konjac gum powder as a stabilizer in ice cream was also developed not only to investigate ice cream quality but also to reduce ingredient costs. Konjac gum is considered safe (GRAS) and contains D-glucose and D-mannose. It is a neutral polysaccharide and has unique properties such as thickening, gelling, texturizing, and water binding. The viscosity of ice cream prepared with 0.3% konjac gum was higher than that of the other treatments (0.1% and 0.5% konjac gum adding) . XanthanWu et al. evaluated meltdown behavior by varying levels of stabilizers (polysorbate 80). Additionally, Wu et al. determined in their study that the fat destabilization value of ice cream samples changed from 8.8 to 73.2%, and the mix viscosity ranged between 0.0220 and 0.2904 Pa.s. Ice cream viscosity and fat destabilization increased as stabilizer levels increased . EmulsifThe goal of this study was to look into the potential use of beeswax oleogels in ice cream. When the amount of saturated fat in the ice cream is reduced, it becomes softer. We aimed to show that various emulsifier and stabilizer systems can be used to produce ice cream with the desired texture and edible qualities. The other goal of this study is to produce alternative ice creams that are less expensive than other methods but are completely liquid oil-based and do not contain trans-fat. They were then tested to see if this structured ice cream production method was suitable for forming the desired texture and consistency in ice cream. Since ice cream typically has a high fat content (10–14%), preparing low-fat diet versions by replacing milk fat with oleogel will help make ice cream healthier by reducing the amount of fat. Ice cream production technology aims to produce alternative and innovative ice cream at a lower cost than other methods. Since salep has been used as a stabilizer for a long time in Turkey, its amount is gradually decreasing. For this reason, konjac gum will be an important alternative in place of salep as a stabilizer in ice cream production.Some physicochemical properties of milk, cream, sunflower oil, and oleogel used in ice cream making are given in The results of physicochemical analysis of the mix and ice cream samples are given in p < 0.01) than that of milk fat. The dry matter ratios noted by Sulejmani and Demiri . Ice cream samples were made with 1500 g milk (3% milk fat or non-fat), 40 g non-fat milk powder, 90 g raw cream (50% milk fat) or oleogel, 300 g sucrose, 15 g stabilizers salep or konjac gum (KG) and 8 g emulsifiers’ monoglyceride (MG) or Palsgaard (PG). At A, D, G, and J samples, emulsifiers were not used (NE) as .Ice cream samples were made in an ice cream machine  and hardened in a deep freezer at −20 °C. All analyses in the study were initiated in duplicate.Dry matter, ash, fat and oil content and specific weights of milk, cream, and oleogel were measured according to these methods . Specifia*, and b* values of the samples were recorded in the reading performed with the device. According to this, the L* value is brightness in daylight , the a* value is green-red , The b* value represents blue-yellow  [Dry matter, ash, fat and oil content, and specific gravity were determined according to the methods given by the researchers . Titrata yellow) . Viscosi yellow) . Additio yellow) ; first d yellow) .A technique based on the process was used to calculate the fat destabilization index in ice cream and the melted ice cream samples. For this, ice cream mix (1 g) was diluted 500 times with distilled, deionized water. The samples were then centrifuged for 5 min at 1000 rpm. Absorbance was measured 10 min later at 540 nm on spectrophotometer . Fat destabilization was calculated as (A mix− A ice cream) × 100/A mix .The basis of the fatty acid analysis is to determine the methylated fatty acids esters given to the gas chromatography according to the differences in the retention times and to determine their ratio as% according to the obtained peaks. Fatty Acid analyses of ice cream samples were made using the methods proposed by Sukhija and Palmquist .Lipid extraction was performed for free fatty acids (FFA) using the modified procedures by Deeth et al. . Two graFatty acids methyl esters (FAME) were performed according to the (AOAC 996.01) Satchithanandam et al.  procedur®, Bellefonte, PA, USA) was used as a standard. FAME peaks were identified by comparing results against the retention times and chain lengths specified in the FAME standard [The fatty acid methyl ester (FAME) composition was analyzed using a Thermo Electronic  gas chromatograph-MS (Model TRACE GC Ultra) equipped with (Model AS-3000-Thermo Electronic Company) autosampler using a silica capillary column  and a flame ionization detector (FID).The injection temperature was programmed between 10 °C and 220 °C at 2 °C/min. Helium was used as the carrier gas with a flow rate of 1.6 mL/min and a partition ratio of 30: 1. Analysis of the fatty acids profile was determined as fatty acid methyl esters. Next, 300-μL of thawed and thoroughly mixed sample was taken in an 11-mL screw-capped test tube. Samples dissolved in 3 mL of isooctane and 2 mL of 1.5 N sodium methoxide were then vortexed at a higher rate for exactly 3 min and allowed to separate for 5 min, and the supernatant was injected into the gas chromatograph. The injection split ratio was 1:80. A Lab Solution computer program was used to control the GC/FID system, and the standard FAME mix  at 20 × magnification. Images were transferred to a computer using a digital camera  and analyzed with the Image-Pro Plus 6.0  software program .Ice cream samples were placed in special 100 mL odor-free containers with glass lids and presented to the panelists in a randomly coded manner at regular intervals. While the panelists were performing sensory analysis, water was placed in 250 mL glass bottle containers to clean their mouths before moving on to the next sample. Sensory evaluations were made by considering color and appearance, texture and consistency, taste and smell, sweetness and gummy structure and general acceptability.Sensory evaluation was performed in a spaced seating arrangement in a room at an appropriate temperature (25 °C). Ice cream samples (–10 °C) were presented to the panelists. Samples were evaluated by panelists of 10 experts  from the Department of Food Engineering using scoring tables from 1 to 9.Each panelist was an experienced individual trained and informed about sensory analysis methodology .p < 0.01 level.The trial was set up as 2 (addition of oleogel and cream) × 2 (addition of stabilizer) × 3 (addition of emulsifier) according to a 2 × 2 × 3 fully random factorial trial plan. The experiment was set up according to a 2 × 2 × 3 full random factorial experiment plan, as 2 (addition of oleogel and cream) × 2 (addition of stabilizer) × 3 (addition of emulsifier). The experiment was carried out in three replications. Two-way analysis of variance (TWO-way ANOVA) was used for the determination the differences between the ice cream samples and the effects of storage. Accordingly, the statistical analysis software program SPSS version 15.0  was used. The significant data as a result of analysis of variance (ANOVA) were tested according to the Duncan multiple comparison test at


Borrelia miyamotoi disease. These spirochetes each encode several surface-localized lipoproteins that bind components of the human complement system to evade host immunity. One borrelial lipoprotein, BBK32, protects the Lyme disease spirochete from complement-mediated attack via an alpha helical C-terminal domain that interacts directly with the initiating protease of the classical complement pathway, C1r. In addition, the B. miyamotoi BBK32 orthologs FbpA and FbpB also inhibit C1r, albeit via distinct recognition mechanisms. The C1r-inhibitory activities of a third ortholog termed FbpC, which is found exclusively in relapsing fever–causing spirochetes, remains unknown. Here, we report the crystal structure of the C-terminal domain of Borrelia hermsii FbpC to a limiting resolution of 1.5 Å. We used surface plasmon resonance and assays of complement function to demonstrate that FbpC retains potent BBK32-like anticomplement activities. Based on the structure of FbpC, we hypothesized that conformational dynamics of the complement inhibitory domains of borrelial C1r inhibitors may differ. To test this, we utilized the crystal structures of the C-terminal domains of BBK32, FbpA, FbpB, and FbpC to carry out molecular dynamics simulations, which revealed borrelial C1r inhibitors adopt energetically favored open and closed states defined by two functionally critical regions. Taken together, these results advance our understanding of how protein dynamics contribute to the function of bacterial immune evasion proteins and reveal a surprising plasticity in the structures of borrelial C1r inhibitors.Borrelial pathogens are vector-borne etiological agents known to cause Lyme disease, relapsing fever, and  Borreliella (Borrelia) burgdorferi, has become a powerful model system for understanding mechanisms of microbial complement evasion    , 16, 17,rrentis) , 19, androchetes , 22, 23.in vivo and is important for optimal infection in a murine model of Lyme borreliosis   was shown to be critical to both C1r-binding affinity and complement inhibitory activity  , 31, 32.BBK32-C) , 31. Smai.e., BBK32-E324) were identified in a homolog from avian-associated Borrelia garinii  that demonstrated significantly reduced activity against human complement  resulted in a protein that lacked measurable C1r binding and caused a complete loss of serum resistance in a serum-sensitive Borrelia burgdorferi background . The precise inhibitory functions of the K2 site are less clear. However, the K2 site, which is ∼25 Å away from the K1 site, interfaces with nonactive site residues on a surface loop of C1r known as the B-loop. The importance of the K2 site is accentuated by the BBK32-K327A mutant, which exhibits an ∼150-fold decrease in C1r-binding affinity and significantly reduced complement inhibitory activity  resulted in a protein that lost all C1r binding and complement inhibitory activity, which provided independent validation of the importance of these conserved sites. Interestingly, FbpB bound only to the active form of C1r, and the crystal structure of the C-terminal region of FbpB revealed significant deviations in the secondary structure of the FbpB K2 site relative to both BBK32 and FbpA  and is instead replaced with a small insertion loop that interrupts the fifth alpha helix. Furthermore, several residues on this K2 site loop in FbpB are apparently flexible, as evidenced by a lack of interpretable electron density for these residues in crystallographic experiments of FbpB  and C4b-binding protein (C4BP)  . This prn (C4BP) . In a fouricatae . While nronectin . Finallynfection . This prnfection . Collectnfection , 33, 34.B. hermsii strain HS1, termed FbpC-C hereafter. Our biochemical, microbiological, and biophysical studies demonstrate that B. hermsii FbpC is a potent inhibitor of the CP of human complement. Surprisingly, however, the high-resolution crystal structure of FbpC-C exhibited a striking difference in the conformation of the K1 site compared to BBK32, FbpA, and FbpB. Furthermore, a disordered stretch of eight residues in the FbpC-C K1 site suggested that this site may be dynamic. Based on these observations, we tested the hypothesis that protein dynamics play a previously unrecognized role in the structure and function of borrelial C1r inhibitors by carrying out detailed analysis of 1 μs molecular dynamics (MD) simulations in tandem with biochemical and biophysical assays utilizing site-directed mutants of FbpC-C.In this study, we report the structural and functional characterization of the C-terminal region of a representative FbpC family member from B. burgdorferi BBK32-C (PDB ID: 6N1L), B. miyamotoi FbpA-C (PDB ID: 7RPR), and B. miyamotoi FbpB-C (PDB ID: 7RPS) have all been previously reported  that diffracted to 1.50 Å limiting resolution .The crystal structures of the C-terminal complement inhibitory domains of reported , 31. HowphaFold2 . Iteratii.e., R248). A homologous arginine residue  was also shown to be important for C1r binding by FbpA-C, and FbpB-C presents R309 in a structurally similar position to both BBK32-C and FbpA-C  takes the place of the key K1 site arginine residues noted above for BBK32-C/FbpA-C/FbpB-C , 3120, 3C/FbpB-C .B). While the AlphaFold2 model was characterized by overall very high confidence, as judged by predicted local distance difference test (pLDDT) ≥90  . The second notable feature of this region of the FbpC-C crystal structure was the lack of electron density corresponding to the residues that connect the first and second alpha helix (residues M247-RMSGHST254) (A and S1A). Flexible regions of proteins are often not visualized in crystal structures  ≥90 , we noteGHST254) A and S1Aructures , and thuructures , 31.i.e., K327)  is also important for C1r binding and inhibition  that interrupts the fifth alpha helix , 31i.e.,A and B)   . Like BB50 of 8.6 nM in an ELISA-based assay . We note that the weaker IC50 of 320 nM observed in this assay compared to the more sensitive C4b deposition ELISA assay is consistent with our previous observations of BBK32-C inhibitory activity (B. burgdorferi strain (B. burgdorferi B314) that lacks all linear plasmids and is highly sensitive to human serum-based complement-mediated killing , indicating that FbpC is sufficient to protect this strain from human complement-mediated bacteriolysis. FbpC-C also was unable to inhibit the lectin pathway of complement at concentrations up to 1 μM, indicating that FbpC-C is CP-specific like BBK32-C . Ta. Ta50 of6N1L, 7RPR, 7RPS, 8EC3), we next set out to utilize MD simulations to test the hypothesis that BBK32 and/or Fbps are structurally dynamic at either the K1 site, K2 site, or both functional sites. This hypothesis was primarily formed from the crystallographic studies showing an increase in temperature factors in the K1 sites of all four proteins (A), a lack of K1 site electron density for FbpC-C, and a lack of K2 site electron density for FbpB-C  and is a measure of the stability of a given simulation (A–D). BBK32-C demonstrated the lowest RMSD values, ranging across the three simulations at 1.70 to 2.52 Å, followed by FbpA-C (2.36–3.04 Å) and FbpB-C (2.25–2.60 Å). FbpC-C RMSD values were relatively higher over the other three inhibitors, ranging between 3.29 and 3.81 Å, suggesting an increase in overall structural flexibility of FbpC-C compared to BBK32-C, FbpA-C, and FbpB-C.RMSD describes the average displacement of atoms at a snapshot in time relative to a reference frame  is dynamic in all four inhibitors with relatively increased dynamics observed for FbpC-C. While relatively less flexible in all proteins compared to the K1 site, the K2 site in FbpB-C is more dynamic than BBK32-C, FbpA-C, and FbpC-C, which is consistent with the lack of electron density for this loop in the prior FbpB-C crystal structure . RMSF is conventionally calculated for groups of atoms that comprise a residue and is compared to the initial starting structure . Residuetructure .Figure 4B) (B).To further investigate the dynamics of the K1 and K2 sites in borrelial C1r inhibitors, we carried out all atom normal mode analysis B 44, 45, 45B (44B) , 51, 52.B) B.B) and, taken together, suggests that borrelial C1r inhibitor proteins may utilize a highly flexible K1 site and anticorrelated motion at the K2 site to generate variation in the distance between the two functional sites.To further investigate the relative conformations of the K1 and K2 sites in these proteins, we carried out dynamic cross correlation matrix (DCCM) analysis of the MD simulations. DCCM measures positional displacements of the Cα atoms at a sample rate of 100 ps, which provides a graphical representation of time-correlated motions of residues within a given protein , 55, 56.Fig. S5i.e., K1 and K2 sites)—which are separated in space by ∼25 to 30 Å—are sampling a larger range of conformations in their unbound forms, particularly for FbpC-C. To test this, we first defined the distance between the K1 and K2 sites in each protein by measuring the distance between two representative residues from each site using the Cα atom  or a distal atom from the side chain as follows: (i) BBK32-C; K1: R248, K2: K327; (ii) FbpA-C; K1: R264, K2: K343; and (iii) FbpB-C; K1: R309, K2: N402. As noted above, the FbpC sequence differs in this area from the other three proteins and a sequence alignment predicts that H252 is analogous to the K1 site arginine residues from BBK32-C, FbpA-C, and FbpB-C. However, we noted that an arginine residue is also present on the flexible K1 site loop in FbpC-C   . Thus, wA–E). Distances between K1 and K2 sites in BBK32-C ranged from 22 to 30 Å  and 12 to 35 Å . We then projected two-dimensional free energy surfaces using main chain and side chain K1-K2 site distances as order parameters . Similar results were obtained for FbpA-C with the most favorable conformations leading to K1-K2 distances between 25 to 27 Å (main chain) and 20 to 28 Å (side chain) . FbpB-C produces a similar shape of the free energy surface plot and indicates that the most favorable K1-K2 site distances range from 19 to 22 Å (main chain) and 22 to 26 Å (side chain) . These results reveal that in the unbound form, BBK32-C, FbpA-C, and FbpB-C sample a similar range of energetically favorable states that modulate the relative positions of the two major C1r-binding surfaces (A–C), which presumably adopt a fixed distance in the bound state.Using these measures, we first analyzed how K1-K2 site distances change during the simulation , A–E. Dirameters , 58. ThiF). The largest basin, represented by the cyan structure in Fig. S6F, involves energetically favorable conformations that produce K1-K2 site distances of 18 to 20 Å (main chain) and 18 to 23 Å (side chain). A more closed conformation (represented by the green structure) and more open conformation (represented by the yellow structure) are also apparent . A distinct free energy surface is also seen when H252 is used to measure the K1-K2 site distance in FbpC-C (D). Two energy minima are observed that collectively span 21 to 30 Å (main chain) and 14 to 25 Å (side chain). This suggests that FbpC-C adopts energetically favorable conformations that produce a wide range of distances of the K1 and K2 sites. Also, in contrast to the other three inhibitors, the secondary structure of the K1 site undergoes considerable rearrangement during the time course of the simulation. As an example, the conformation of the more closed state  involves an unwinding of alpha helix 2. Likewise, the helical structure of the K1 site observed in the more open state  differs from that found in the central energy basin .Unlike the other energy contours, FbpC-C exhibits three clear energy minima when R248 is used in the measurement of the K1-K2 site distance F. The lan FbpC-C D. Two ened state D, green en state D, yellowgy basin D, cyan.i.e.: R248 or H252) was involved in C1r interactions based on sequence and structural alignments  than WT FbpC-C (KD = 0.3 nM) and even more so for zymogen C1r where a KD could not be reliably determined (A and B). Consistent with this C1r-binding deficit, CP-specific ELISAs showed that the H252A mutation had a pronounced effect on CP inhibition with an IC50 value of 300 nM, compared to 8.6 nM for WT FbpC-C  . We also asked what the importance of each the K1 site residues was for FbpC-C’s activity in a functional bacterial cell-based assay , we exogenously added in each recombinant protein: FbpC-C WT, FbpC-R248A, or FbpC-H252A. While FbpC-R248A exhibited a reduction in its ability to protect B. burgdorferi B314 from serum-mediated killing relative to its WT counterpart, FbpC-H252A was even further impaired in its ability to prevent killing . Both strains were compared to B. burgdorferi B314 expressing FbpC (pAP20) as a positive control and vector-only B314 pBBE22luc as a negative control for complement resistance. These data highlight that both residues affect binding and inhibition of C1r to various degrees; however, mutation of H252 had the largest effect. Taken together, these results underscore the importance that dynamic residues of the K1 site play in the overall complement-inhibitory properties of FbpC-C.FbpC-R248A bound zymogen and active C1r with respective termined , A and Bed assay , 30. UsiB. burgdorferi BBK32 and the orthologous Fbp proteins of relapsing fever and B. miyamotoi spirochetes . We cannot rule out the influence of the crystallization process/conditions in stabilizing the conformation observed for the FbpC-C K1 site, but we note that BBK32-C, FbpA-C, and FbpB-C were all crystallized such that the K1 sites were in a more closed conformation. Despite the structural differences for FbpC-C, we showed that it retains high affinity for human C1r and inhibits human complement in a manner similar to BBK32 and B. miyamotoi FbpA   —providesA) , 40. ThiA)  and 4 (3A) , 40. PreA)  and aideA) . Regardi devices . To our  devices , 68 and B). Free energy surface analysis showed that BBK32 and Fbp proteins adopt energetically favorable “open” and “closed” states defined by the variation in distance between the functional K1 and K2 sites  as has been reported for FbpB . While we have shown here that FbpC does not inhibit a closely related complement pathway  were subcloned into pT7HMT  corresponding to residues 212 to 374, were obtained from IDT Technologies gBlock Gene Fragment Service. Residue numbering is based on UniProt numbering (G9BXS5). Each FbpC-C DNA fragment was subcloned into pT7HMT as previously described , a monoclonal antibody to BBK32 , or B. burgdorferi strain B31 FlaB , washed, and then incubated with a 1:10,000 dilution of goat anti-mouse IgG-HRP (Thermo Fisher Scientific). The blots were processed to visualize the immune complexes as previously described (Western immunoblots were conducted to detect FbpC and BBK32 from constructs produced in B. hermsii FbpC in strain B314 was determined using proteinase K accessibility assays as described (luc (vector control) and B314-pCD100 (produces BBK32). A monoclonal antibody to BBK32 was used to confirm that bbk32 was not being expressed and to test whether this BBK32-specific monoclonal cross reacted with FbpC when produced in B314-pAP2.Surface expression of escribed  with a pRecombinant FbpC-C, FbpC-C R248A, and FbpC-C H252A proteins were expressed and purified as in . BrieflyB. hermsii FbpC were generated by immunizing female C57BL/6 mice intradermally with 25 μg of purified FbpC-C in an equal volume of PBS and TiterMax Gold Adjuvant (Sigma Aldrich) as outlined .Polyclonal antibodies reactive with outlined . Two wee212–374 was concentrated to 8 mg/ml in 10 mM Tris (pH 8.0), 50 mM NaCl buffer. Crystals of FbpC-C were obtained by vapor diffusion of sitting drops at 25 °C. Crystals grew in a condition containing 0.2 M MgCl2, 0.1 M Bis-Tris (pH 5.5), 25% PEG 3,350, and generally appeared in 3 days. Crystals were harvested and cryoprotected by supplementing the crystallization buffer with 10% glycerol.FbpC-C1 and contained a single copy within the asymmetric unit. Initial phases for FbpC-C were obtained by molecular replacement using the predicted FbpC-C structure from AlphaFold2 (www.rcsb.org/) under the PDB ID 8EC3. A description of crystal cell constants, diffraction data quality, and properties of the final refined model can be found in www.pymol.org/).Monochromatic X-ray diffraction data were collected at 1.0 Å wavelength using beamline BM-22 of the Advanced Photon Source . Diffraction data were integrated, scaled, and reduced using the HKL2000 software suite  and assephaFold2  35). Fo. Fo1 andphaFold2 , 78, 79 phaFold2 . The finphaFold2 , 78, 79.B. hermsii FbpC212–374  via standard amine coupling as before (KD) and kinetic rates were obtained from the resulting kinetic fits obtained using Biacore T200 Evaluation Software .SPR was performed on a Biacore T200 instrument at 25 °C using a flowrate of 30 μl/min. HBS-T running buffer was used (10 mM Hepes (pH 7.3), 140 mM NaCl, and 0.005% Tween 20), which was supplemented with 5 mM CaCls before , 32. The2, 0.5 mM MgCl2, and 0.1% gelatin). A primary mouse antibody α-C4c (Quidel) was diluted to 1:10,000 followed by a secondary goat α-mouse antibody conjugated to horseradish peroxidase (Thermo Fisher Scientific) at a 1:3000 dilution to determine complement activation by measurement of C4b deposition. Data were obtained in at least triplicate and normalized to a well containing serum only (100% complement activation) and background subtracted using a negative control with buffer containing no serum. Plates were washed three times between each step with tris-buffered saline . An EnSight Multimode Plate Reader (PerkinElmer) was used to read the absorbance values at 450 nm for the reaction. Half-maximal inhibitory concentration values (IC50) were obtained using the nonlinear variable slope regression fit from GraphPad Prism version 9.5 (https://www.graphpad.com/).ELISA-based assays that utilized IgM as a specific activator of the CP or mannan as an activator for the lectin pathway as described in , 32, 82 B. hermsii FbpC-C WT, H252A, or R248A protected erythrocytes from complement-mediated hemolysis. A serial dilution series of FbpC-C proteins ranging from 1.95 nM to 4 μM were incubated with buffer-exchanged presensitized sheep erythrocytes (Complement Technology) and 2% NHS (Innovative Research). After a 1h incubation at 37 °C, the reaction was clarified via centrifugation at 500 × g and the absorbance read using an EnSight Multimode Plate Reader (PerkinElmer) at 412 nm. Data were obtained in triplicate and normalized using a positive control with no inhibitor present that represented the 100% hemolysis at 37 °C in 1 h. Background hemolysis was subtracted out using a negative control with only buffer and red blood cells.A CP hemolysis assay was performed as previously described , 30 to dB. burgdorferi strain B314 producing B. hermsii FbpC (pAP2), as well as the vector-only control B314 pBBE22luc, were grown at 32 °C in 1% CO2 in complete BSK-II media with kanamycin at 300 μg/ml to early- to mid-log phase. The cells were then diluted in complete BSK-II media to a final cell density of 1 × 106 cells/ml, then incubated for 1.5 h in 15% NHS (Complement Technology) or equivalent heat inactivated serum as a positive control for survival. Cell survival was assessed by dark field microscopy based on cell motility and membrane damage or lysis.Assays were performed as outlined previously , 31, 32.B. hermsii FbpC-C, and either the R248A mutation or H252A mutation, to rescue serum-sensitive B. burgdorferi B314 pBBE22luc was determined as in . Residues not visualized in the final crystal structures due to poor electron density were first modeled with MODELLER v9.1 . OPLS-AA force fields . A total of 24,698, 24,283, 23,751, and 33,073 water molecules were used to solvate BBK32-C, FbpA-C, FbpB-C, and FbpC-C systems, respectively. An application of periodic boundary conditions of 10 Å from the edge of the cubic box of dimensions 91.3, 90.8, 90.2, and 100.5 Å, respectively, was implemented. Charge neutralization of the simulation systems for BBK32-C, FbpA-C, FbpB-C, and FbpC-C were undertaken by replacing water molecules with 5 Cl-, 3 Cl-, 1 Cl-, and 2 Cl-ions, respectively. The final systems to be simulated were thus comprised of 76,440 atoms for BBK32-C, 75,355 atoms for FbpA-C, 73,926 atoms for FbpB-C, and 101,858 atoms for FbpC-C. Bond lengths for each of these systems were constrained by LINCS algorithm  is measured in K (298 K) at which all the simulations were performed  determines the time interval over which the Cij elements are calculated. Positive Cij values result from backbone atom motions between residues i and j that are in the same direction along a given spatial coordinate, while negative Cij values result from backbone atom motions between residues i and j that are opposite in direction along a given spatial coordinate. To normalize such positional fluctuation, all the MD snapshot frames extracted for calculations were superposed to a common frame of reference .A time scale is associated with each Chttp://www.wwpdb.org/, 8EC3. All other data are contained within the manuscript.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Infectious bronchitis virus (IBV) belongs to the gamma-coronavirus genus of Coronaviridae and causes serious infectious diseases in the poultry industry. However, only a few IBV strains can infect avian passage cell lines, seriously hindering the progress of basic research on IBV pathogenesis. Whereas IBV field strains can replicate in tracheal ring organ culture (TOC) without any previous adaptation in chicken embryos or primary cells. In this study, to investigate the potential use of TOC as an in vitro infection model for the study of IBV-host interaction, we first established a chicken embryo TOC culture system and carried out an investigation on the IBV replication kinetics in the system. We found that the selected strains of the IBV GI-1, GI-7, GI-13, GI-19, and GI-22 genotypes could successfully replicate in TOC and bring about damage to the infected trachea. Next, we identified host proteins of the chicken embryo trachea that interact with the IBV S1 protein by immunoprecipitation and protein mass spectrometry. A total of 127 candidate proteins were initially identified with major involvement in cell adhesion pathways and apoptosis- and autophagy-related pathways. The heat shock protein 70 (HSP70) was selected for further investigation in the interaction with IBV viral proteins. Our results showed that HSP70 interacted with IBV S1 in both TOC and CEK cells, whereas HSP70 overexpression inhibited viral replication. This study indicates that TOC is a good system for the elucidation of IBV-host interactions and HSP70 is a potential host antiviral factor. Coronaviruses (CoVs) are classified into four genera based on phylogeny: alpha-CoV, beta-CoV, gamma-CoV, and delta-CoV. Avian infectious bronchitis virus (IBV) is a major species coronavirus in the gamma-CoV group, causing infectious bronchitis (IB) and impairing the respiratory, reproductive, digestive, and even nervous systems of poultry of all ages and different species . Due to As one of the members of the CoVs, IBV possesses a single-stranded, positive-sense RNA genome, which comprises two overlapping open reading frames (ORFs) encoding polyproteins 1a and 1ab, the main structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N). The viral S protein is the major surface protein of IBV, which is cleaved into S1 and S2 subunits by the host serine protease furin . The S1 Tracheal ring organ culture (TOC) is a commonly used and reliable method for isolating and culturing IBV, as an alternative to embryonated chicken eggs. IBV field isolates that do not replicate in primary cell culture can replicate and proliferate in TOC without previous adaptation in embryonated chicken eggs . With thThe interaction between viral and host proteins is one of the most important routes for viruses to successfully infect host cells and carry out replication. Heat shock proteins (HSPs), including heat shock protein 70 (HSP70), are a large family of chaperone proteins found in most eukaryotes and bacteria. HSPs are responsible for the correct folding of proteins, protecting cells from stress, stimulating the production of immune and inflammatory factors, and regulating the host immune response. During the course of viral infection, it is utilized by the virus to complete the viral life cycle at all stages and to adapt to and escape the host’s antiviral response [HSP70 is a receptor or coreceptor of various viruses, including dengue virus, Japanese encephalitis virus, and rotavirus ,9,10. To2 atmosphere. Chicken embryonic kidney (CEK) cells were prepared as previously described [IBV reference strains Beaudette (GI-1) and M41 (GI-1) as well as field isolates GX191219 (GI-7), QD191227 (GI-13), HF200702 (GI-19), and YC181031 (GI-22) used in this study were stored in our laboratory and were genotyped according to the typing method established by Valastro et al. . Vero an2. After culturing for 12–24 h, tracheal rings with active ciliary beating were used for infection.Tracheas were removed from 18 to 20 days old embryonated chicken eggs and placed into petri dishes containing DMEM. Fats and surrounding connective tissues were discarded as much as possible, and the lumen was rinsed 5–10 times using a syringe filled with DMEM. The cleaned tracheas were transversely sectioned into 0.5–1 mm tracheal rings with small sharp scissors. The rings were then washed twice with culture medium and placed into 24-well plates with 4 rings in each well containing 0.5 mL TOC medium (DMEM supplemented with 5% FBS and 0.1% antibiotic (containing 100 i.u./mL penicillin and 100 ig/mL streptomycin)) and cultured in a 37 ℃ cell incubator with 5% CO50 of the IBV virus strain was measured using tracheal rings with vigorous beating cilia after a 24 h preparation. Serial dilutions of IBV-infected chicken embryo allantoic fluid ranging from 10−1 to 10−10 were inoculated into the tracheal rings in the wells of the 24-well plates, with 5 wells for each dilution, followed by incubation at 37 °C with 5% CO2 for 1 h. Next, after 3 washes with PBS, the tracheal rings were cultured with TOC medium in the incubator for 144 h. During this period, the culture medium was renewed every 48 h. The samples were scored as infection-positive when 70% ciliostasis was observed. The calculation of TOC-ID50 was conducted according to the Reed–Muench method as follows: distance ratio = (positive rate above 50% − 50%)/(positive rate above 50% − positive rate below 50%) and lgTOC-ID50 = logarithm of the highest antibody dilution above 50% positive rate + distance ratio × logarithm of the dilution factor.The TOC-ID50, and uninfected TOCs were used as controls. Successful infection was confirmed by RT-PCR using one set of primers targeting the IBV N gene (IBV-N-F: 5′-GATGGTAATTTCCGTTGGGA-3′/IBV-N-R: 5′-CATTGTTCCTCTCCTCATCT-3′). Briefly, 3 tracheal rings in 500 μL PBS were processed using a tissue crusher for 1 min, followed by 3 rounds of repeated freezing and thawing, and the supernatant was subjected to total RNA extraction using TRIzol reagent. The RNA was then transcribed into cDNA according to the instructions provided by the Reverse Transcription Kit . PCR was performed in a 25 μL reaction containing 12.5 μL of 2× PCR mixture, 0.5 μL of each primer (10 μM), and 1.5 μL of cDNA (10 ng/μL), then subjected to the amplification programs at 95 °C for 5 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, an extension at 72 °C for 30 s, and a final extension of 72 °C for 10 min. Real-time qRCR (RT-qPCR) was performed to detect the relative abundance of IBV-N transcripts using the ChamQ Universal SYBR qPCR master mix  in a LightCycler 96 sequence detector system . A grading system of 0 (100% ciliostasis) to 4 (0% ciliostasis) was used to score the ciliary activity of the TOC. The activity of control tracheal rings at 12 hpi was recorded at the highest level of “4”, whereas the scores in the other groups ranged from 0 to 4 at the different infection time points, respectively. For further analysis, the dynamics of IBV infection in TOC were monitored via 6 samples for each time point with 3 rings for HE, IHC, and IFA assays, with the remaining 3 were used for Western blot analysis.Freshly prepared TOCs with vigorously beating cilia were infected with different IBV strains at a dose of 1000 TOC-IDThree tracheal rings in each tube were washed once with 4% paraformaldehyde (PFA), then kept in 1 mL 4% PFA and sent to Nanjing Freethinking Biotechnology Co., Ltd.  to perform hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) assay. The membrane proteins from IBV YC181031 infected TOCs were extracted using the Membrane Protein Extraction Kit  following the manufacturer’s protocol. Membrane protein extracts were immunoprecipitated (IP) with mAb 1H1 against IBV S1 protein and a mAb against mouse IgG . The precipitated proteins were detected by silver staining and Western blot analyses using anti-S1 and anti-HSP70 antibodies. Next, 293T cells were cotransfected with pCMV-Flag-N-HSP70/Myc-N-BD-S1, and the Flag-tagged HSP70 was immunoprecipitated with Flag antibodies and captured by Sepharose A/G beads. The precipitated proteins were then detected by Western blot analysis using anti-Flag antibodies and mAb 1H1 to IBV S1.TOC lysates and immunoprecipitated proteins were separated using SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking with 5% skim milk, the membranes were incubated with the indicated primary antibodies at 4 °C overnight. After three washes with PBS, the blots were incubated with HRP-labeled anti-mouse/rabbit IgG at 4 °C for 1 h. Protein bands were then visualized using an enhanced chemiluminescence reagent and imaged using an AI680 Imager .The polyacrylamide gel was fixed with a 4:1:5 ratio of ethanol, glacial acetic acid, and water at room temperature for 2 h and then washed thrice with water. After incubation with a sensitizing solution containing sodium acetate, sodium thiosulfate, and absolute ethanol, the gel was transferred to the silver stain solution and incubated in the dark at room temperature for 30 min. Finally, a color developer containing anhydrous sodium carbonate, formaldehyde, and sodium thiosulfate was used and incubated in the dark with the gel. The reaction was terminated by adding 5% glacial acetic acid. The stained bands were imaged using an ImageScanner III instrument, and the bands of interest were excised from the gel for liquid chromatography-mass spectrometry (LC-MS) analysis.Three independent immunoprecipitation experiments to identify the host proteins interacting with IBV S1 were performed. After silver staining detection, specific bands in the IBV S1 mAb group as compared to the control IgG group were collected from three independent gels and sent for commercial mass spectrometry (LC-MS/MS) analysis carried out by Shanghai Applied Protein Technology Company. Protein searches were carried out using Mascot 2.2 software. Protein identification was performed using the following criteria: (a) trypsin-digested peptides with 2 max missed cleavages allowed, (b) proteomics tools: 3.1.6, (c) >1 unique peptides, (d) filter by score ≥20. Proteins found in the respective negative control samples were eliminated from the dataset to remove nonspecific interactions. Proteins represented by at least two unique peptides were considered for further analysis, and the proteins in the negative control were excluded.https://david.ncifcrf.gov/home.jsp, accessed on 17 March 2021) under the three major categories of biological processes (BO), molecular functions (MF), and cell components (CC).Candidate proteins from LC–MS/MS analysis were subjected to GO annotation and KEGG analysis by using the DAVID website (The plasmid pCMV-Flag-N-HSP70 was transfected into Vero cells using the jetPRIME kit (Polyplus). The cells were infected with the IBV Beaudette strain (MOI = 1) 24 h after transfection and incubated at 37 °C for 12 h, 24 h, and 36 h, respectively. The cells were harvested after three washes with PBS and subjected to Western blot analysis.50 values and IBV infection. As calculated by the Reed–Muench method, the TOC-ID50 values of the M41 reference strain and the GX191219, QD191227, HF200702, and YC181031 field isolates were 10−5.0/mL, 10−5.8/mL, 10−6.56/mL, 10−6.77/mL, and 10−7.5/mL, respectively.The ciliary activity of TOCs was inhibited during preparation but recovered gradually upon culturing at 37 °C. After 24 h, the ciliary activity of TOCs remained at a high level and could be used to determine virus TOC-ID50 were used to infect tracheal ring samples, and ciliostasis of the TOCs was observed under a microscope over a 144-h period. As shown in Five selected IBV strains including M41 and four field isolates  at a dose of 1000 TOC-IDTo further investigate the histological lesions imposed on the trachea upon IBV infection, HE staining was performed on IBV YC181031 infected TOCs at different infection time points. Histopathological examination revealed that the epithelial cells of the tracheas in the infected group suffered degeneration, with increased intracellular vacuoles observed at 12 hpi. At 24 hpi, the morphology of the epithelial cells of the infected TOCs was not as clear as that of the control group, and necrosis and exfoliation of epithelial cells were observed in the infected group. Cilia in the infected tracheas were lessened at 24 hpi and completely disappeared by 96 hpi; edema of the lamina propria occurred by 48 hpi and lymphocyte infiltration was noticed at 96 hpi in infected group A. In conThus far, the TOC approach has rarely been used in the study of IBV-host interactions. To identify potential host proteins binding to IBV S1 in the TOC, we first checked viral protein expression during infection. As shown by Western blot analysis, the expression level of S1, N, and M proteins of IBV YC181031 in TOC peaked at 24–36 hpi A; therefThe membrane proteins obtained from the lysate of TOC infected with YC181031 at 36 hpi were extracted and subjected to immunoprecipitation assays with mAb 1H1 against IBV S1 protein and anti-mouse IgG. IBV S1 subunits around 100 kDa from the immunoprecipitated fragment were detected by Western blot analysis with mAb 1H1 to IBV S1, but not detected in control IgG B. Two spTo establish the host cell proteins or pathways that have been enriched in the IBV S1-TOC protein interaction network, we performed gene annotation and analysis on the biological processes, molecular functions, and cell components of the candidate proteins. A large number of biological processes, such as glomerular visceral epithelial cell development, regulation of myoblast differentiation, and peroxisome-proliferator activated receptor binding, were enriched. In addition, adherens junction, transcriptional coactivator activity, and chloride channel inhibitor activity were enriched under molecular functional categories, whereas apical junction complexes, chromosome telomere regions, apical plasma membranes, and flotillin complexes were enriched under cell component classifications .In order to further predict the cellular metabolic and signal transduction pathways of candidate host proteins interacting with IBV S1 protein, we conducted a pathway enrichment analysis using the KEGG database. The pathways targeted by the candidate proteins analyzed by KEGG are presented in Immunoprecipitation assay was performed with mAb 1H1 against the IBV S1 protein in S1 and HSP70 cotransfected 293T cells and YC181031 infected TOCs. As shown in To preliminarily understand the effects of HSP70 on IBV replication, the IBV Beaudette strain was used to infect HSP70-overexpressing Vero cells. Infected cells were collected at 12, 24, and 36 hpi and subjected to Western blot analysis and RT-qPCR. As shown in In this study, one IBV reference strain M41 and four isolate strains  were used to infect the tracheal rings of chicken embryos, respectively. The degree of ciliostasis was used to evaluate the activity of tracheal tissues infected with IBV [The selected virus replicated in epithelial cells very well. As shown by IHC analysis, almost every epithelial cell was infected by the virus at 12 hpi B, while Using IHC analysis, we clearly noticed epithelial cells stained by IBV mAb at 12 hpi, after which the number of stained cells decreased B. HoweveGO and KEGG analyses were performed on the identified candidate proteins. Enrichment analysis results showed that the host proteins interacting with the S1 protein are closely related to cell adhesion and connection, protein metabolism, and transport processes and participate in receptor interaction, apoptosis, and autophagy signaling pathways . Among tA previous study identified heat shock protein 70 (HSP70) from the lungs and kidneys of IBV-infected chickens as a binding-associated protein to IBV S1 of the SCZJ3 strain, and infection of CEK cells by SCZJ3 was found to be inhibited by anti-HSP70 antibodies, indicating that HSP70 is a part of the IBV receptor complex . HSP70 tWe further investigated the effects of HSP70 on IBV replication by using knockdown and overexpression assays. However, we could not detect clear changes in HSP70 expression by transient transfection of HSP70 interference or overexpression plasmids using the TOC system (data not shown). Additional factors present in TOC may influence the knockdown and overexpression of host genes. In addition, due to the high inherent expression level of native HSP70 in the trachea organ, it may not be easy to interfere with its expression or to observe the effects of HSP70 overexpression upon virus infection. Further fine-tuning or modification of reaction conditions is required in order to use the TOC model to study the functions of host proteins on IBV infection.Instead of using TOC, in Vero cells, we observed that HSP70 overexpression inhibited IBV Beaudette growth, indicating that HSP70 might be a potential host anti-viral factor for IBV infection . This reIn conclusion, we evaluated TOCs as an ex vivo culture model for infections with various IBV strains and carried out a study on IBV virus-host protein interaction. We found that all five selected different genotype IBV strains could replicate in the epithelial cells of the trachea of embryonated chicken eggs and gave rise to ciliostasis, loss of cilia, and cellular necrosis. A total of 127 predicted IBV S1-interacting host proteins were identified in infected TOCs, including interesting candidates potentially related to IBV invasion. Among them, HSP70 was further confirmed to interact with IBV S1 protein in both TOCs and transfected cells, an interaction that has potential use in antiviral therapy.


To quantify 5-HT2A receptor availability, the binding potential (BPND) was derived using the basis function implementation of the simplified reference tissue model, with the cerebellum as the reference region. The participants’ aggression levels were assessed using the Buss–Perry Aggression Questionnaire. The voxel-based correlation analysis with age and sex as covariates revealed that the total aggression score was significantly positively correlated with [11C]MDL100907 BPND in the right middle temporal gyrus (MTG) pole, left fusiform gyrus (FUSI), right parahippocampal gyrus, and right hippocampus. The physical aggression subscale score had significant positive correlations with [11C]MDL100907 BPND in the left olfactory cortex, left orbital superior frontal gyrus (SFG), right anterior cingulate and paracingulate gyri, left orbitomedial SFG, left gyrus rectus, left MTG, left inferior temporal gyrus, and left angular gyrus. The verbal aggression subscale score showed significant positive correlations with [11C]MDL100907 BPND in the bilateral SFG, right medial SFG, left FUSI, and right MTG pole. Overall, our findings suggest the possibility of positive correlations between aggression traits and in vivo 5-HT2A receptor availability in healthy individuals. Future research should incorporate multimodal neuroimaging to investigate the downstream effects of 5-HT2A receptor-mediated signaling and integrate molecular and systems-level information in relation to aggression traits.Serotonergic neurotransmission has been associated with aggression in several psychiatric disorders. Human aggression is a continuum of traits, ranging from normal to pathological phenomena. However, the individual differences in serotonergic neurotransmission and their relationships with aggression traits in healthy individuals remain unclear. In this study, we explored the relationship between 5-HT2A receptor availability in vivo and aggression traits in healthy participants. Thirty-three healthy participants underwent 3-Tesla magnetic resonance imaging and positron emission tomography (PET) with [ Serotonergic neurotransmission has been implicated in impulsive and aggressive behaviors in animals and humans ,2,3,4. MAlthough aggressive behaviors are a common feature of borderline PD, antisocial PD, and several other psychiatric disorders, even healthy individuals can be at increased risk of such behaviors under certain conditions, such as when hatred and resentment are fueled by a variety of factors, e.g., economic inequality , interpeTo date, individual differences in serotonergic neurotransmission in vivo and their relationships with aggression in healthy participant groups have been investigated in a few PET molecular imaging studies of 5-HT1A ,22, 5-HTAggression traits are multidimensional and composed of several subcomponents, such as physical aggression, verbal aggression, anger, and hostility . These t11C]MDL100907, a radioligand that provides both high selectivity for 5-HT2A receptors and a high signal-to-noise ratio, and aggression traits  in healthy individuals. We chose 5-HT2A receptor availability based on the findings of a recent postmortem study on comprehensive serotonergic markers in subjects with impulsive and aggressive behaviors MDL100907 binding potential with respect to non-displaceable compartment (BPND) in the temporal pole of the right middle temporal gyrus (MTG) (uncorrected p < 0.0001), left fusiform gyrus (FUSI) (uncorrected p < 0.0001), right parahippocampal gyrus (PHG) (uncorrected p = 0.0001), and right hippocampus (HIP) (uncorrected p = 0.0001). The physical aggression subscale score had significant positive correlations with [11C]MDL100907 BPND in the left olfactory cortex (uncorrected p < 0.0001), orbital part of the left superior frontal gyrus (SFG) (uncorrected p < 0.0001), right anterior cingulate and paracingulate gyri (uncorrected p < 0.0001), medial orbital part of the left SFG (uncorrected p = 0.0001), left gyrus rectus (GR) (uncorrected p = 0.0003), left inferior temporal gyrus (ITG) (uncorrected p < 0.0001), left MTG (uncorrected p < 0.0001), and left angular gyrus (AG) (uncorrected p = 0.0001). The verbal aggression subscale score was significantly positively correlated with [11C]MDL100907 BPND in the medial part of the right SFG (uncorrected p < 0.0001), left FUSI -corrected p = 0.028), bilateral SFG , and temporal pole of the right MTG (uncorrected p < 0.0001). The scores on the anger and hostility subscales did not show significant correlations with [11C]MDL100907 BPND at the significance threshold. Details of the results are presented in 11C]MDL100907 BPND values in the clusters with statistical significance are shown in The voxel-based correlation analysis with age and sex as covariates revealed that the total aggression score was significantly positively correlated with [11C]MDL100907 BPND in automated anatomical labeling (AAL) [11C]MDL100907 BPND in the temporal pole of the right MTG  and right HIP . The physical aggression subscale score had positive correlations with [11C]MDL100907 BPND in the orbital part of the left SFG , medial orbital part of the left SFG , and left GR . The verbal aggression subscale score also showed positive correlations with [11C]MDL100907 BPND in the temporal pole of the right MTG . No other correlations were observed at p < 0.05. Details of these results are shown in We further conducted a supplementary region-of-interest (ROI)-based correlation analysis between the aggression scores and MDL100907 binding does not compete with endogenous serotonin in humans [11C]MDL100907) are unlikely, the higher BPND in our study can generally be interpreted as being driven mainly by a higher Bmax and vice versa. In contrast, high postsynaptic 5-HT2A receptor binding may reflect compensatory upregulation owing to chronically low 5-HT2A receptor occupancy by low endogenous serotonin MDL100907 BPND was quantified using the basis function implementation of the simplified reference tissue model (SRTM); however, a two-tissue compartment model (2TCM) with arterial input function is optimal [11C]MDL100907 BPND values were obtained using the basis function implementation of the SRTM with the cerebellum as the reference region devoid of specific binding, as previously suggested for this tracer MDL100907 . A computed tomography-based transmission scan was conducted for attenuation correction prior to [11C]MDL100907 injection. After a bolus injection of 688.0 ± 52.5 MBq [11C]MDL100907 with an average molar activity of 65.0 ± 32.9 GBq/μmol  with MDL100907 BPND in the right calcarine gyrus and right cuneus  compared to males  . Further0 voxels . A voxelto males .11C]MDL100907 BPND in the brain regions of healthy individuals, we performed a voxel-based partial correlation analysis with age and sex as covariates using SPM12. As our study was exploratory in nature, without a priori hypotheses, significant results were defined at a peak-level threshold of uncorrected p < 0.001 with an extent threshold of 20 voxels, which has been used as a statistical significance threshold in previous PET studies MDL100907 PET, in specific cerebral regions in healthy individuals. Future research should incorporate multimodal neuroimaging to investigate the downstream effects of 5-HT2A receptor-mediated signaling and integrate molecular- and system-level information in relation to aggression traits.Our findings suggest the possibility of positive correlations between aggression traits and in vivo 5-HT2A receptor availability, as measured using [


Target site resistance to herbicides that inhibit protoporphyrinogen IX oxidase  has been described mainly in broadleaf weeds based on mutations in the gene designated protoporphyrinogen oxidase 2 (PPO2) and in one monocot weed species in protoporphyrinogen oxidase 1 (PPO1). To control PPO target site resistant weeds in future it is important to design new PPO‐inhibiting herbicides that can control problematic weeds expressing mutant PPO enzymes. In this study, we assessed the efficacy of a new triazinone‐type inhibitor, trifludimoxazin, to inhibit PPO2 enzymes carrying target site mutations in comparison with three widely used PPO‐inhibiting herbicides.Amaranthus spp. PPO2 enzymes were expressed in Escherichia coli, purified and measured biochemically for activity and inhibition kinetics, and used for complementation experiments in an E. coli hemG mutant that lacks the corresponding microbial PPO gene function. In addition, we used ectopic expression in Arabidopsis and structural PPO protein modeling to support the enzyme inhibition study. The generated data strongly suggest that trifludimoxazin is a strong inhibitor both at the enzyme level and in transgenics Arabidopsis ectopically expressing PPO2 target site mutations.Mutated Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.Trifludimoxazin is a potent PPO‐inhibiting herbicide that inhibits various PPO2 enzymes carrying target site mutations and could be used as a chemical‐based control strategy to mitigate the widespread occurrence of PPO target site resistance as well as weeds that have evolved resistance to other herbicide mode of actions. © 2022 BASF SE and The Authors.  Amaranthus plants may contain more than one PPO target site mutation. Trifludimoxazin is effective in controlling PPO enzymes carrying double mutations.Protoporphyrinogen IX oxidase (PPO)‐inhibiting herbicide resistance is mainly endowed by target site mutations.  Theoretically, hundreds of targets for herbicides that affect the growth and development of plants exist, yet the number of targets of commercial relevance for herbicide products is limited, as shown in Herbicide Resistance Action Committee (HRAC’s) mode of action and chemistry overview poster.PPO inhibitors impair the PPO enzyme,Upon PPO enzyme inhibition, the PPO substrate protoporphyrinogen is exported outside the chloroplast and mitochondria, and strongly accumulates in the cytoplasm where nonspecific cytoplasmic oxidases convert it to protoporphyrin.The PPO enzyme is a particularly good target with respect to druggability. Structurally diverse chemistry classes are still being identified, allowing the synthesis of many new inhibitors.Eleusine indica, a PPO1 target site mutation was found to mediate resistance specifically to oxadiazon.Commercially used PPO active ingredients applied in this study  show little selectivity, with strong and fast control of broadleaf weeds and lesser control of grasses. The difference in sensitivity between monocots and dicots was described early on as being caused by physiological differences.During the past 15 years, PPO inhibitors have gained importance in the control of weeds resistant to glyphosate and ALS (acetolactate synthase) inhibitors.E. indica mentioned above. One PPO2 target site mutation has been identified in A. artemisiifolia, A. tuberculatus and A. palmeri as R128G, R128M, R128L (or R98L in A. artemisiifolia).A. tuberculatus and A. palmeri was also shown to confer resistance to several PPO‐inhibiting herbicides including lactofen and fomesafen.−18 compared with approximately 10−9, respectivelyA. tuberculatus), was reported to confer a high level of resistance to PPO inhibitors.A. tuberculatus and/or A. palmeri populations collected in several US states.Nontarget site resistance has been reported for fomesafenAmaranthus PPO2 double target site mutants toward trifludimoxazin, and the resistance profiles are supported by analysis of major binding properties and in silico substrate binding to mutants.In this study, we show the resistance profile of various 22.1A. tuberculatus, PPO2 variant proteins and the enzymatic assay to determine protein activity and median inhibitory concentration (IC50) is provided in Rangani et al.Complete description of the expression and purification of 2.2To investigate the effect of mutations on trifludimoxazin binding, we started with in‐house PPO2 structures co‐crystalized with trifludimoxazin as the basis for modeling. The crystal structure of the wild‐type, and of the ΔG210 variant, is available. Other PPO2 co‐crystal structures were used as a basis to model other herbicides, for example fomesafen into the binding site. After superposition on crystallographically observed binding modes, local minimization of the herbicides was performed. To examine the effect of the mutations G399A and R128G on ligand binding, the wild‐type crystal structure was modified by virtually mutating G399 and R128 to alanine and glycine, respectively. Local minimization of the complex was performed after introducing these mutations. For molecular modeling, the program MOE was used .2.32.3.1Arabidopsis thaliana seeds  were sown in a substrate composed of GS90 soil + 5% sand. Seeds were subjected to stratification for 5 days at 4°C, followed by a short‐day growth period of 10 days . After that, plants were transplanted into 8 × 8 cm pots filled with GS90 soil and cultivated under the same conditions for 14 days, under long‐day growth conditions  and maintained until seed harvest. Plants were fertilized with 0.3% Hakaphos Blau (15–10–15 NPK) twice a week until flowering. Relative humidity was not controlled, but kept between 40% and 70% during all growth stages, except during stratification.2.3.2ppx‐2 L from A. tuberculatus were inserted into a RTP6557 transformation vector, which was then inserted into Agrobacterium tumefaciens strain C58C1pMP90. The gene insert also included an acetolactate synthase herbicide‐resistance trait as a selectable marker to identify transformed Arabidopsis seedlings. This would ensure that plants eventually tested for resistance to PPO‐inhibiting herbicides all expressed the transgene.To prepare the transgene, wild‐type and mutant variants of 2.3.3Agrobacterium culture containing the plasmid was prepared a day before dipping by inoculating 1 ml of glycerol stock into 250 ml of YEB medium  plus the appropriate antibiotic. Bacteria were cultured for 12 h at 28°C with continuous agitation at 150 rpm. The next day, after adjusting the Agrobacterium culture density to OD600 = 1.0 (with YEB medium), the culture was collected by centrifugation at 1600 g for 10 min and resuspended in 150 ml of infiltration medium composed of 2.2 g L−1 Murashige & Skoog (MS) medium, 50 g L−1 sucrose, 0.5 g L−1 MES hydrate, 10 μl L−1 BAP . The pH was then adjusted to 5.7–5.8.The 2.3.4Plants with immature floral buds were dipped in the bacterial suspension for 10 s after adding 75 μl of Silwet‐L77 per 150 ml of infiltration medium to a jar. After dipping, plants were kept overnight in a cabinet under high humidity and low light intensity and were grown under long‐day conditions until maturity. When siliques turned yellow, plants were placed in paper bags to collect the seeds. T1 seeds were then transferred to falcon tubes and stored at 4°C.2.3.5Arabidopsis plants. Sowing and stratification were performed as described previously.−1] and three concentrations of trifludimoxazin  foliar‐applied when plants reached the ten‐leaf stage, using a spray chamber calibrated to deliver 375 L ha−1 of spray solution. Herbicide efficacy was visually assessed after 7 days from herbicide treatments. Herbicides spray solution and empty control contain the adjuvant DASH HC (DASH) containing 349 g/l oil (fatty acid esters) and 209 g/l alkoxylated alcohols‐phosphate esters, reference ID no. 30059102, BASF.After 14 or more days of storage at 4°C, T1 seeds were sown to select putative transgenic 2.4600 of 0.05 in the assay. The herbicide starting concentration in the assay was 10−5 m with ten further dilutions 1:5 in 60% dimethyl sulfoxide. Ten microliters of herbicide was added to 190 μl of prepared cells to give a total assay volume of 200 μl. The assay was prepared in a clear and sterile 96‐well flat bottom plate with a lid. Absorbance was measured every 30 min at 600 nm (OD600) for 24 h at 37°C with shaking.The PPO2 R128(X) ΔG210 mutants of interest were cloned in the pET21 plasmid (ampicillin selection). The SAS38X cells were transformed by electroporation and plated on Luria–Bertani ampicillin agar plates. Colonies were visible after 48 h at 37°C. An overnight culture picked from a single colony was diluted with Luria–Bertani ampicillin for a starting OD2.4.1Vmax). The Vmax value, the maximum enzyme velocity under saturated substrate condition, of the positive control (cells only with dimethyl sulfoxide) is equivalent to 0% inhibition. Calculation of the percent inhibition values for each herbicide in all ten dilutions determined the IC50.valueThe maximum slope was determined using at least eight measuring points  were calculated. IC50 is the concentration of a given herbicide that causes 50% in vitro protein inhibition and reflects the herbicide inhibition potency. Therefore, high IC50 values indicate a poor inhibition effect, whereas low IC50 values indicate strong inhibitory potency  ΔG210 double‐mutant enzymes ranged from 10−7 to 10−6 for R128C ΔG210, R128E ΔG210, R128H ΔG210, R128I ΔG210, R128L ΔG210, R128Q ΔG210 and R128T ΔG210. IC50 values greater than 10−5 (which was assessed as highly resistant) were found for R128A ΔG210, R128M ΔG210 and R128N ΔG210. The other nine variants were inactive or below the detection limit of the assay. The inhibition potency of saflufenacil and fomesafen towards R128(X) ΔG210 mutants was low (IC50 below 10−5 m) in most cases, indicating poor inhibition potency. Thus, lactofen appears to be a better inhibitor than saflufenacil and fomesafen in inhibiting PPO2 enzymes carrying R128(X) ΔG210 double mutations. R128D ΔG210, R128S ΔG210, R128Y ΔG210, R128F ΔG210, R128P ΔG210, R128W ΔG210, R128V ΔG210 and R128G ΔG210 are less likely to occur in planta because these double‐mutant enzymes were found to be inactive in vitro and are likely to impose a significant plant fitness cost. The IC50 values for trifludimoxazin were much lower than those found for saflufenacil, fomesafen and lactofen, and ranged from 10−9 to 10−7 m, showing that this inhibitor has a greater ability to inhibit R128(X) ΔG210 mutants at the enzyme level. To confirm this further, the plant response (ED50) values of lactofen, saflufenacil and trifludimoxazin were determined using an in vivo growth‐based system that utilizes the hemG E. coli strain complemented with Amaranthus PPO2. In agreement with the in vitro enzyme assay results, trifludimoxazin showed the highest inhibition potency against heterologously expressed R128(X) ΔG210 variants .The ICts Table . The E. 3.3A. palmeri and A. tuberculatus was predicted by in silico mutating each nucleotide of the triplet that encodes R128. Single mutation on the triplet is more likely to occur than double or triple mutations. R128 is encoded by an AGG codon in A. palmeri and AGA codon in A. tuberculatus  ΔG210 target site mutations, the mutants were ectopically expressed in 3.5A detailed study of the binding mode of trifludimoxazin, for which internal protein crystal structures were created, reveals several features that explain the high affinity to plant PPO. Together with molecular modeling, it could be explained how trifludimoxazin inhibits the major PPO2 target site mutations including double‐mutant combinations. Trifludimoxazin binds in the same substrate‐binding pocket as protoporphyrinogen, right next to the co‐factor flavin adenine dinucleotide (FAD). The pocket is deeply buried in the protein. Beta‐sheet secondary structures form the posterior wall of the pocket, which is covered from above by an α‐helical structure and defined from below by the co‐factor Figure . Triflud4PPO‐inhibiting herbicides are effective tools to control a broad spectrum of weeds, including those that have evolved resistance to glyphosate.Mutations on Phe381 and Val383 of PPO2 were predicted to not affect trifludimoxazin binding because their side chains are directed away from the binding site  ΔG210 double‐mutant enzyme variants was reduced more by trifludimoxazin than by saflufenacil (Fig. 35S:: promoter, which would result in a greater level of tolerance than the endogenous PPO2 promoter.In this article, we show the inhibitory capability of trifludimoxazin toward PPO2 enzymes carrying target site mutations. At the PPO2 enzyme level, trifludimoxazin was shown to inhibit R128L, R128G R128M and ΔG210 to a greater extent than other competitor benchmarked products, including saflufenacil, butafenacil, lactofen and fomesafen.Arabidopsis is commonly used to screen the effect of herbicide‐resistance genes,Amaranthus. Therefore, controlling Arabidopsis plants at 1× and 2× the field rates of trifludimoxazin does not fully guarantee control of Amaranthus expressing PPO2 double mutations. However, all the above indications point out that trifludimoxazin has greater inhibition capability than the other PPO inhibitors tested towards PPO2 enzymes carrying target site mutations. This might be translated into greater biomass control of resistant weeds in the field.E. coli mutant hemG for lactofen, trifludimoxazin and saflufenacil (Tables Lolium rigidum. PPO1 target site resistance was recently found in the grass Eleusine indica.At the enzyme level, trifludimoxazin exhibited a lower resistance factor when compared with saflufenacil, lactofen and fomesafen. This was also confirmed in an in vivo growth‐based assay that utilizes the l Tables  and 4. Ul Tables . At the Mitigating herbicide resistance often requires a diversified strategy that does not rely only on the use of chemicals. Although trifludimoxazin is probably the most potent PPO‐inhibiting herbicide on the market, sole reliance on this herbicide may impose strong selective pressure that promotes resistance. Therefore, use of trifludimoxazin should always be combined with other control strategies to preserve its efficacy in the long term and slow the evolution of resistance.5Compared with the other PPO inhibitors evaluated, trifludimoxazin provides greater inhibition of PPO2 enzyme with mutations related to herbicide resistance, even when they are combined. Consequently, trifludimoxazin has potential to combat existing weed populations with target site resistance to PPO inhibitors. Further molecular structure‐based design of PPO‐inhibiting herbicides is needed in future to ensure the management of existing target site mutations and possible upcoming additional mutations derived from selective pressure. Based on trifludimoxazin, we could show how to use binding groups to a β‐wall amino acid residue, which could be used in future to adapt further inhibitors in the PPO active site. However, technologies like DNA‐encoded library screening offer further ways to screen billions of molecules to find new herbicides. Although the target site resistance profile was assessed, we cannot exclude at this stage that nontarget site mechanisms might reduce the efficacy of PPO‐inhibiting herbicides, including trifludimoxazin.Authors affiliated with BASF contributed to the planning and implementation of research activities. All other authors declare no conflict of interest.