D/Gi‐IC50). Ligands with high intrinsic efficacy  stimulated biphasic (Gi‐Gs) concentration responses, however for ligands with low intrinsic efficacy , responses were monophasic (Gi‐only). ERK1/2‐phosphorylation responses appeared to be Gi‐mediated. For Gs‐mediated responses to be observed, both a system with high receptor reserve and high agonist intrinsic efficacy were required. From the Gi‐mediated efficacy ratio, the degree of Gs‐coupling could be predicted. The clinical relevance and precise receptor conformational changes that occur, given the structural diversity of compounds with high intrinsic efficacy, remains to be determined. Comparison with α1 and β1/β2‐adrenoceptors demonstrated subclass affinity selectivity for some compounds  whilst e.g., oxymetazoline had high affinity for both α2A and α1A‐subtypes, compared to all others. Some compounds had subclass selectivity due to selective intrinsic efficacy . A detailed knowledge of these agonist characteristics is vital for improving computer‐based deep‐learning and drug design.α2‐adrenoceptors, , are Gi‐coupled receptors. Central activation of brain α2A and α2C‐adrenoceptors is the main site for α2‐agonist mediated clinical responses in hypertension, ADHD, muscle spasm and ITU management of sedation, reduction in opiate requirements, nausea and delirium. However, despite having the same Gi‐potency in functional assays, some α2‐agonists also stimulate Gs‐responses whilst others do not. This was investigated. Agonist responses to 49 different α‐agonists were studied  in CHO cells stably expressing the human α2A, α2B or α2C‐adrenoceptor, enabling ligand intrinsic efficacy to be determined (binding K  CHOChinese hamster ovaryPBSphosphate buffered salinePDBUphorbol 12,13‐dibutyratePTXpertussis toxinsfmserum free media = DMEM/F12 containing 2 mM L‐glutamine1α2‐adrenoceptors, comprising α2A, α2B and α2C‐subtypes, are Gi‐coupled G‐protein coupled receptors (GPCRs) expressed in heart, blood vessels and kidney  and are highly expressed throughout, including the prefrontal cortex and locus coeruleus.A broad range of α2‐agonists exist with different pharmacological and physicochemical properties and clinical uses. Dexmedetomidine is one of the most potent α2‐agonists to dateα2C‐adrenoceptors and appear particularly prevalent in the striatum and hippocampus.α2B‐adrenoceptors appear very minor in brain.The remaining 10% of brain α2‐adrenoceptors are α2‐adrenoceptors have been extensively studied. The original studies were restricted to using different tissue preparations ‐ human platelet, colonic adenocarcinoma or rat cortex for α2A, neonatal rat lung for α2B and opossum kidney for α2C; e.g.,Agonist drugs  have 2 important properties – affinity (ability to bind to a receptor) and intrinsic efficacy , 3H‐CGP12177, Microscint 20 and Ultima Gold XR scintillation fluid were from PerkinElmer . Foetal calf serum was from Gibco (Thermo‐Fisher), Lipofectamine and OPTIMEM were from Life Technologies, Thermo‐Fisher, Massachusetts USA. All other cell culture reagents were from Sigma Chemicals . Even though they are the same compound, brimonidine and UK14304 were purchased from different suppliers so are reported separately throughout. Medetomidine (racemate) and the active isomer dexmedetomidine were also purchased separately so reported separately.All compounds, together with the supplier and catalogue number are given in alphabetical order in Supplementary Data Table 2.22: 95% air atmosphere. Cells were always grown in the absence of any antibiotics. Mycoplasma contamination has intermittently been monitored within the laboratory (negative) but cell lines were not tested routinely with each experiment.CHO‐K1 (RIDD: CVCL_0214) stably transfected with a CRE‐SPAP reporter gene and the human α2A‐adrenoceptor (CHO‐α2A), human α2B‐adrenoceptor (CHO‐α2B) or human α2C‐adrenoceptor (CHO‐α2C) were used2.32). After 5 h, all drugs and media were removed, 40 μL sfm was added to each well and the cells incubated for a further hour at 37°C before being incubated at 65°C for 30 min (to destroy any endogenous phosphatases), cooled to 37°C, 100 μL 5 mM pNPP in diethanolamine buffer added to each well and incubated at 37°C until the yellow color developed before being read on a Dynatech MRX plate reader at 405 nm.CRE‐SPAP production was measured as in.2.43H‐adenine (0.5 mL per well) for 2 h at 37°C (5% CO2). The 3H‐adenine was removed, each well washed by the addition and removal of 1 mL sfm, then 0.5 mL sfm containing 100 μM IBMX added to each well. Agonist in 5 μL (diluted in sfm) was added to triplicate wells and incubated for 10 min at 37°C. Where used, forskolin (10 μM) was then added to the wells, and plates incubated for 5 h at 37°C (5% CO2). The reaction was terminated by the addition of 50 μL concentrated HCl per well, the plates were then frozen, thawed and 3H‐cAMP separated from other 3H‐nucleotides by Dowex and alumina column chromatography, with each column being corrected for efficiency by comparison with 14C‐cAMP recovery as previously described.Cells were grown to confluence in 48‐well clear plates. Cells were pre‐labeled by incubation with 2 μCi/mL 2.5Extracellular‐signal‐regulated kinases (ERK1/2) activation was measured using a Surefire Alphascreen pERK1/2 kit. Cells were grown to confluence in 96‐well clear plates and double serum starved by washing the cells twice with 100 μL sfm before incubating in a further (third) 100 μL sfm for 24 h. Agonists in 20 μL sfm were added to the well  and incubated for 2–4 min (at 37°C). Reagents were then removed, 20 μL lysis buffer added to each well and ERK1/2‐phosphorylation measured using the Alphascreen kit as per manufacturer's instructions. After a minimum of 2 h in the dark, the plates were read on an EnVision plate reader using standard Alphascreen settings. Basal and maximum ERK1/2‐phosphorylation  was measured in each plate.2.63H‐rauwolscine (diluted in sfm) and incubated for 2 h at 37°C . The media and all drugs were then removed from the wells, the cells washed twice by the addition and removed of 2 × 200 μL 4°C PBS. Cells were inspected under a light microscope to ensure they were still adherent after the wash, and 100 μL Microscint 20 was then added to each well. Total binding and non‐specific binding (determined by the presence of 10 μM RX821002) was defined in every plate. Radioligand concentrations were determined from taking the average of triplicate 50 μL samples of each 3H‐rauwolscine concentration used and counted on a PerkinElmer TriCarb Scintillation counter.The affinity of the agonists was assessed using the whole cell binding and is identical to that used to determine the affinity of agonists at the α1‐adrenoceptors2.72.7.150 is the concentration of agonist that produces 50% of the maximal response.Many agonist responses were best described by a one‐site sigmoidal agonist concentration‐response curve. These were fitted to the data using the following equation with Graphpad Prism 7:2.7.250 is the concentration of agonist that inhibits 50% of the response to forskolin (Gi‐coupled response), EC50 is the concentration of agonist that caused a half maximal stimulation (Gs‐coupled response) and SMAX is the maximum stimulation of this Gs‐coupled‐component.Many concentration response curves clearly contained two components – an inhibitory response followed by a stimulatory response, thus a two‐site analysis was performed using the following equation:2.7.3KD values were calculated from the parallel shift of the agonist concentration responses in the presence of a fixed concentration of antagonist using the following equation:Antagonist In experiments where three different fixed concentrations of the same antagonist were used, Schild plots were constructed using the following equation:2.7.4KD value is stated, increasing concentrations of agonist fully inhibited the specific binding of 3H‐rauwolscine (unless otherwise annotated in the tables). The following equation was then fitted to the data using Graphpad Prism 7 and the IC50 was determined as the concentration required to inhibit 50% of the specific binding.50 is the concentration at which half of the specific binding of 3H‐rauwolscine has been inhibited.In all cases where a 50 value, the known concentration of 3H‐rauwolscine and the known KD3H‐rauwolscine (determined from saturation binding),KD value  was calculated using the Cheng‐Prusoff equation:3H‐rauwolscine binding. Where no inhibition of radioligand binding was seen, even with maximum concentration of competing ligand possible, “no binding” is given in the tables. Where the inhibition produced by the maximum concentration of the competing ligand was 50% or less, an IC50 could not be determined and thus a KD value not calculated. This is shown in the tables as IC50 > top concentration used (i.e. IC50 > 100 μM means that 100 μM inhibited some but less than 50% of the specific binding). In cases where the competing ligand caused a substantial  inhibition of specific binding, an IC50 value was determined by extrapolating the curve to non‐specific levels and assuming that a greater concentration would have resulted in 100% inhibition. These values are given as apparent KD values in the tables.From the ICn in the text refers to the number of separate experiments. Affinity selectivity ratios are given as a ratio of the KD values for the different receptors, and intrinsic efficacy is given as efficacy ratios determined from KD/IC50.All data are presented as mean ± SEM of triplicate determinations and http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY,Key protein targets and ligands in this article are hyperlinked to corresponding entries in 33.150–8.94 ± 0.05, n = 26), followed by a stimulation of CRE‐SPAP production at higher concentrations , and stimulatory response in the absence of forskolin , very similar to the CRE‐SPAP responses  and the stimulatory brimonidine response .KD −8.48 ± 0.13, n = 15; Figure n = 14 or in the presence of PTX . Finally no response was seen to brimonidine in cells without the transfected receptor (see later).To confirm that both parts of these responses were occurring via the α2A‐adrenoceptor, the α2‐selective antagonist yohimbine was used to inhibit the response. Increasing concentrations of yohimbine caused a rightward shift of both the inhibitory , however, there was a direct relationship between the receptor expression level and the ability to induce a Gs‐stimulatory response (both in the presence and absence of forskolin). As shown in supplementary Figure 3.3Not all agonists stimulated a biphasic response. Moxonidine stimulated a clear biphasic CRE‐SPAP production response, whilst naphazoline, despite a similar potency for the Gi‐component, did not Figure 3. Further3.450 . From the KD values obtained and the IC50 value from the Gi‐inhibition of CRE‐SPAP production, an efficacy ratio (KD/IC50)KD −6.37 ± 0.07, n = 5, Figure 50 (−8.94) giving an intrinsic efficacy ratio of 2.57. This was similar for moxonidine (2.49). However, the efficacy ratio for naphazoline was only 0.78. The ligands in Table KD from binding. However given the close correlation between IC50 and ERK1/2‐phosphorylation EC50, similar results would have occurred from using efficacy ratio calculated using the ERK1/2‐phosphorylation as the functional response.Affinity measurements were made from 3.6KD values of −7.62 ± 0.14 and −7.66 ± 0.03 respectively .50 −8.19 ± 0.11, log EC50 −6.56 ± 0.08, n = 7) and absence  of forskolin and the ERK1/2‐phosphorylation response closely resembled the IC50 obtained from Gi‐inhibition . Once again, the ERK1/2‐phosphorylation response  stimulated potent responses in the CHO‐β1 and CHO‐β2 cells, however significant agonist responses and measureable affinity were also seen in response to a few α‐agonists e.g., etilefrine, metaraminol, phenylephrine and methoxamine , whose responses were considerably less potent and much smaller in amplitude than those seen in the α2A cell lines. The bromocriptine responses in Tables There were no CRE‐SPAP responses to any of the agonist ligands examined the parental CHO‐CRE‐SPAP cell line , either in the presence (looking for Gi responses) or absence (looking for Gs responses) of forskolin  closely mirroring the Gi‐inhibitory response. Correlation plots of IC50 (Gi‐mediated 5 h CRE‐SPAP inhibition) vs EC50 (2–4 min ERK1/2‐phosphorylation) give straight lines  to be studied separately and a measure of intrinsic efficacy (efficacy ratio) obtained. For brimonidine and moxonidine, the efficacy ratio was high (log 2.57 and 2.48 respectively), suggesting few receptors need occupying to stimulate agonist responses (i.e. the compounds had high intrinsic efficacy). Naphazoline had a lower efficacy ratio at 0.78 (lower intrinsic efficacy). Table This explains others' findings Eason et al.,Thus (1) high receptor reserve and (2) high ligand intrinsic efficacy are both required for observation of Gs‐coupling. What remains unknown, is how higher ligand concentrations induce a different conformational state that alters receptor‐G‐protein coupling, nor whether this phenomenon is relevant in native tissues or clinical responses. Interestingly, dexmedetomidine exhibits a biphasic blood pressure response in people, with low dose infusions reducing blood pressure and high dose infusions increasing blood pressure.The α2B‐adrenoceptor cell line has very high receptor expression, with biphasic responses and substantial Gs‐stimulation with many agonists. ERK1/2‐phosphorylation mirrored the Gi‐inhibitory CRE‐SPAP component Figure  and the The α2C‐adrenoceptor cell line had a lower receptor expression and although agonists inhibited both CRE‐SPAP and cAMP responses (Gi), no Gs‐responses were seen  similar to comparisons from human colonic adenocarcinoma cells (α2A), neonatal rat lung (α2B) and opossum kidney cells (α2C)KD −7.27), and related xylometazoline, also have high α1A‐adrenoceptor affinity  and others having lower efficacy . However, there are some differences . However, it has 1000‐fold higher α1A‐affinity than for any other α‐adrenoceptor, giving rise to more potent α1A functional responses. A61603 is an affinity‐selective α1A‐agonist. Interestingly at α2A‐adrenoceptors, A61603 was the only compound where the Gs‐response was lower than predicted from Gi‐potency and intrinsic efficacy. The reason is unknown, although the binding was so poor that affinity (and efficacy ratio) could not be accurately established.Perhaps more interesting is the comparison between α1 and α2‐subtypes. Dexmedetomidine has 100‐fold higher affinity for α2 than α1‐adrenoceptor subtypes with mid‐range efficacy at all six α‐subtypes, suggesting that affinity is largely driving the higher α2 vs α1‐potency of dexmedetomidine responses. However, brimonidine only has a 10‐fold higher α2 than α1‐affinity but very high α2‐intrinsic efficacy (giving potent responses) and low α1 intrinsic efficacy. The α2‐selectivity of brimonidine appears to be driven more by α2‐selective intrinsic efficacy with less reliance on selective affinity.There are examples of the reverse. R‐phenylephrine, etilefrine, metaraminol and methoxamine have similar affinity across all α‐subtypes but are highly efficacious at α1‐adrenoceptors with low efficacy at α2A and α2C‐subtypes (interestingly α2B is once again a little different). These compounds α1‐selective functional responses are being driven by α1‐selective intrinsic activity, whilst A61603, above, has α1A‐selective affinity.KD/Gi‐IC50), the degree of Gs‐stimulation observed within a given system can be predicted. It remains to be determined whether this Gs‐coupling is clinically relevant and the precise receptor conformational changes that occur, given the structural diversity of compounds with high intrinsic efficacy.In conclusion, both (1) system high receptor reserve and (2) agonist high intrinsic efficacy are required for α2‐Gs‐mediated responses to be observed. From the Gi‐mediated efficacy ratio  whilst others are selective due to intrinsic efficacy (methoxamine/etilefrine:α1 and brimonidine:α2). A detailed knowledge of these agonist characteristics is vital for improving computer‐based drug design.JGB designed the research study. RGWP, JA and JGB performed the research. JGB and JA analyzed the data. JGB wrote the paper.JGB has been on the Scientific Advisory Board for CuraSen Therapeutics since 2019.No animals, human tissue, human volunteers or patients were used in this study.Data S1Click here for additional data file.


Most of the food allergens sensitized via the gastrointestinal tract resist thermal treatments and digestion, particularly digestion by pepsin. Roasted hazelnuts are more commonly consumed than raw ones. Since no studies have characterized gastric digestion protein fragments of raw and roasted hazelnuts nor their IgE binding properties, we compared these aspects of raw and roasted hazelnuts’ gastric digesta obtained by INFOGEST protocol. Their electrophoretically resolved profiles were probed with hazelnut allergic patients’ sera in 1D and 2D immunoblots. Electrophoretic profiles demonstrated pepsin digestion of all hazelnut allergens to varying extents. While 2D immunoblots indicated that roasting slightly reduced allergenicity, IgE ELISA with the pool of sera showed a slight significant (10%) increase in IgE binding in both gastric digesta. Cor a 9 isolated from the raw and roasted hazelnuts, characterized by far and near CD, remained stable after roasting, with preserved IgE reactivity. Its immunoreactivity contribution by inhibitory ELISA was noticeable in raw and roasted hazelnut digesta; its activity was slightly stronger in the roasted preparations. Roasting has a visible impact on proteins; however, it did not affect overall IgE reactivity. Gastric digestion slightly increases the overall IgE reactivity in raw and roasted hazelnuts, and may therefore impact the profiles of allergens and their fragments available to interact with the immune system in the small intestine. Hazelnut allergy prevalence was understudied until recently, despite its clinical importance. Studies have shown that prevalence of hazelnut allergy depends on the patients’ age and their geographical location . Cor a 9Wigotzki et al., (2000) used 1D immunoblot with a defatted hazelnut extract probed with the sera of patients allergic to hazelnut, to show that the IgE-binding activity of the hazelnut allergens was preserved after conventional roasting , decreasing substantially after 15 min of roasting at 185 °C . ProteinIn this study, we applied the INFOGEST 1.0 protocol  on the r−1 protein) and porcine pepsin from gastric mucosa  were purchased from Sigma–Aldrich . The enzyme activities were measured according to the assays detailed by Minekus et al., (2014)  × 100. The results were analyzed in GraphPad Prism 7.0. The IgE binding properties of hazelnut gastric digesta purified from raw and roasted hazelnuts were analyzed using inhibitory ELISA with Cor a 9 as an inhibitor. The liquid phase of non-defatted gastric digesta and their controls were used to coat microtitre plates  with 100 μL per well, incubated overnight at 4 °C in coating buffer  . Figure Overall, when comparing raw and roasted digesta and their respective controls, under all conditions, it seems that roasting affected oral–gastric extraction and consequently also digestion, in terms of creating slightly different electrophoretic profiles. However, no effects on impairing or facilitating the overall extraction process were observed in an acidic environment , skin prick test, histamine release, and enzyme allergosorbent test (EAST), especially in the patient group that was sensitized to birch-pollen-related hazelnut allergens . The expConcerning resistance to oral–gastric digestion, hazelnut allergens were at least partially resistant to oral–gastric digestion according to our results , and the2D profiles of raw and roasted controls and their gastric digesta revealed rather similar proteomic profiles . DiffereThe overall 2D immunoblot outlook map shows that the raw hazelnut control had the highest IgE binding, while IgE reactivity in the roasted digesta was the weakest . Faint IThe basic chain of 11S globulin-like protein (Cor a 9) could easily overlap with a trace of nsLTP (Cor a 8). Therefore, proteomic experiments were conducted to elucidate the nature of this trailing IgE immunoreactivity around pH 9. The presence of Cor a 8, Cor a 9, and Cor a 14 was determined in the control of raw sample, while in its digesta, besides Cor a 8 and Cor a 9, Cor a 11 and Cor a 12 were also found in the low-mass gel region  reported that the thermal processing of Ara h 3 in peanuts resulted in increased IgE binding for every serum tested, compared with the raw peanut counterpart . HoweverSince Cor a 9 remained stable after roasting ,41, an e−1 for protein extracts, and corresponding resulting protein amounts were applied per well in the ratio raw Ct: raw Dg: roast Ct: roast Dg = 1:0.6:1.1:0.7 , because coating saturation plateau was reached in all four preparations, or if it is false due to unequal coating amounts . It is logical to suspect that the effect observed was due to decreased Cor a 9 concentration in the roasted samples  published a study where their subjects of interest were different hazelnut varieties . They sh1D gel A and 1D/It has been shown that the nsLTP is thermo-labile over 90 °C, probably due to the existence of the lipid-binding tunnel , so therIn this study, we investigated the stability and IgE binding of raw and roasted hazelnut allergens when subjected to gastric digestion. The thermal processing, i.e., roasting, visibly affected the hazelnut protein profiles compared with the raw hazelnut profiles. The overall extractable quantities of proteins and peptides were slightly increased by roasting in the acidic environment (raw control vs. roasted control). In addition, the overall extent of the pepsin digestion was not significantly affected when comparing digestion extents between the raw control and raw digesta, and the roasted control and roasted digesta. 1D immunoblots showed differential IgE responses; within the lower mass region (~12 kDa), IgE binding was dominant in the raw controls, while in regions corresponding to Cor a 9 subunits, slightly stronger IgE binding was observed in roasted hazelnut preparations. 2D immunoblots indicated that roasting slightly reduced the allergenicity of control and digesta compared to their raw counterparts. This was opposite to the IgE ELISA with the pool of hazelnut-allergic patients’ sera, which showed a significant 10% increase in IgE binding in both gastric digesta, probably due to the creation of additional smaller peptides during digestion that retained their immunoreactivity. This discrepancy between results most likely arose from the contribution of conformational epitopes in the IgE ELISA tests.Cor a 9 in all preparations (raw and roasted) was a dominant contributor to the allergenicity of the whole hazelnut, implying that the immunoreactivity of Cor a 9 was preserved after roasting and after gastric digestion. In support of this, purified raw and roasted Cor a 9 under physiological conditions had a slightly stronger inhibitory effect on the binding of IgE antibodies from the pool of patients’ sera, in all gastric digestions, compared with controls, meaning that there was a slight decrease in the quantity of available Cor a 9 IgE epitopes after 2 h of simulated gastric digestion. In addition, purified roasted Cor a 9 was an even stronger inhibitor in roasted samples than its raw counterpart against raw hazelnut matrices. In addition to possible epitopes of Cor a 9 due to eventual modifications introduced by roasting, this could also point to a decreased relative abundance of Cor a 9 in the roasted samples. Spectroscopic results and mass spectrometry identifications from the 2D gel basic region are in the support of the latter hypothesis. Namely, the presence of Cor a 9 alongside several other allergens  was shown in basic smears of the raw samples, in contrast to the roasted hazelnut, where only Cor a 8 was identified. In addition, spectroscopic studies performed on Cor a 9 revealed that the secondary structure did not change upon roasting, but instead the tertiary structure was slightly affected. Overall hazelnut allergenicity originates significantly from Cor a 8, as well as from Cor a 9. Further experiments are warranted to elucidate why digesta reacted with greater intensity to the specific antibody for Cor a 8, compared with the controls. To summarize, roasting per se did not affect the overall IgE reactivity of the hazelnut, despite the differential electrophoretic profiles and slightly increased protein extraction in the roasted control. However, gastric digestion per se did affect the overall IgE binding, which was increased significantly in the raw and roasted hazelnut gastric digesta. These results point to the likely protective effect of the lipid-rich food matrix that delayed the extraction of proteins. During the simulated gastric digestion, the lipid-rich food matrix was liberated, further delaying gastrointestinal digestion, which may affect allergen-sensitizing capacity and clinical symptoms.


Pseudomonas strain 1809276 isolated from the urine of a Chinese patient and analyze its integrative and conjugative element (ICE) 1276 formation mechanism.To precisely determine the species of a carbapenem-resistant 1276 were aligned. At the same time, the transfer ability of blaIMP−1 and the antibiotic sensitivity of Pseudomonas juntendi 18091276 were tested.Single-molecule real-time (SMRT) sequencing was carried out on strain 18091276 to obtain the complete chromosome and plasmid (pCN1276) sequences, and average nucleotide identity (ANI) was used for precise species identification. The ICEs in GenBank with the same integrase structure as ICE P. juntendi, and its drug resistance mechanism is the capture of the accA4' gene cassette by the Tn402-like type 1 integron (IntI1-blaIMP−1) to form In1886 before its capture by the ΔTn4662a-carrying ICE 1276. The acquisition of blaIMP−1 confers carbapenem resistance to P. juntendi 18091276.This bacterium was blaIMP−1-carrying ICE 1276, its further integration into the chromosomes, and transposition and recombination of other elements promote bacterial gene accumulation and transmission.The formation of  Pseudomonas sp. is a genus of varied and complex Gram-negative bacteria commonly isolated from soil and water, with a wide host diversity, including animals and plants. It consists of 254 species  divided into three lineages and 13 groups based on the sequences of the 16S rRNA, gyrB, rpoB, and rpoD genes , and then, these genes begin to spread among other bacteria.P. juntendi strain separated from a urine specimen of a patient in China in 2018, which was tentatively identified as P. putida by VITEK2 (bioMerieux), and the strain carried a carbapenem resistance gene blaIMP−1. To our knowledge, this is the first report of clinical strain of P. juntendi carrying blaIMP−1 in a new gene context in China.In this study, we analyzed a Strain 18091276 was isolated from a urine specimen from a patient in a tertiary hospital in northeast China in 2018 and the species of the strain was determined by the part of the sequence of the 16S rRNA gene  of imipenem was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute CLSI,  guidelinde novo by using SMARTdenovo (http://github.com/ruanjue/smartdenovo).Bacterial genomic DNA was extracted from the strain 18091276 using the UltraClean Microbial Kit  and sequenced by a PacBio RSII sequencer . The reads were assembled Rast 2.0 , and ICEberg 2.0 .http://www.ezbiocloud.net/tools/ani). A ≥95% ANI cutoff was used to define bacterial species  program, a neighbor-joining tree reconstruction algorithm, and 1,000 bootstrap replicates  analysis between strain 18091276 and the reference genome , and resuspended in 150 μl of LB. The mixture was spotted on a 1 cm2 hydrophilic nylon membrane filter (Millipore) with a 0.45-μm pore size, which was then placed on an LB agar plate and then incubated for mating at 37°C for 6 h. The cells were recovered from the filter membrane and spotted on LB agar containing 100 μg/ml sodium azide and 4 μg/ml imipenem for selecting carbapenem-resistant E. coli transconjugant.Conjugation experiments were performed as described previously .P. putida, and P. putida, Pseudomonas monteilii, Pseudomonas plecoglossicida, and P. juntendi in the P. putida group was more than 99% and the strain was identified to be in the P. putida group P. juntendi BML3 (GCA_009932375.1), and this strain was confirmed to be P. juntendi  and P. juntendi PSB00036  trade and travel. The identification method at the time was not precise, many P. juntendi isolates were wrongly identified as P. putida or other species in the P. putida group  that was named ICE 1276. ICE 1276 contained attL and attR produced from recombination, a complete integrase gene (int), the F-type IV secretion system responsible for the ICE transfer, the conjugation gene cpl, and rlx responsible for autonomous rolling circle replication  was also found in the backbone region of ICE 1276. crpP was mainly present in the ICEs of Pseudomonas aeruginosa and could increase ciprofloxacin resistance  in plasmid pDK1 . Moreover, it had the same 38 bp inverted repeat sequence (IRs) as Tn4662a, and a 5-bp (AGTAT) directed repeat sequence (DRs) was generated during insertion , the insertion structure of P. juntendi 18091276 also contained a Tn6811 remnant (Tn3 family), a single copy insertion element ISPsfu1 (IS5 family), and a complete In1886 (Tn402-like type 1 integron) carrying blaIMP−1 and aacA4'. However, the res_site_II, res_site_III, tnpR, and tnpA sequences in Tn4662a (ICE 1276) were partially deleted, which should have a Tn4662a-derived structure , but the ANI of LTGT-11-2Z and all Pseudomonas species were lower than 95%. Therefore, LTGT-11-2Z was only identified as Pseudomonas sp. All ICEs were obtained from Pseudomonas sp. chromosomes, and E. coli transconjugants were not obtained after repeated conjugation experiments. Except for the exogenous insertion sites, the remaining structure of the ICEs was nearly identical. Another exception is that a 14.29 kb accessory module was inserted at 74 bp upstream of ssb (single-strand DNA binding protein) in the backbone region of ICE LTGT-11-2Z (plant-derived Pseudomonas sp. LTGT-11-2Z). This ICE seemed to have caused the transfer of large fragments between Pseudomonas species under specific conditions.Until November 2021, only 4 ICEs with the same integrase as ICE Tn4662a is truncated by Tn6811, it is unable to carry out normal replicative transposition  and there were two independent insertion processes. The Tn4662a-drived structure in ICE LTGT-11-2Z .As the downstream region of on Zong, . AlignmeGT-11-2Z  indicateP. aeruginosa strain from Japan . However, as this In1886 ISCfr1 fragment mediated the insertion of strA-strB-aac(3)-IId to the Tn402 recombination site on PA15W-In1886, it resulted in the truncation of the res_site_r1 site  can be found at: All strains were provided by China-Japan Union Hospital, Jilin University. PC, XG, JC, JL, and LZhu conceived, directed, and carried out the study. ML, GL, JJ, YW, XZ, and BJ prepared samples for sequence analysis. JG, SS, and LZhe acquired samples and analyzed the data. All authors have read and approved the final manuscript.Funding for study design, data collection, data generation, and publication costs was provided by the National Science and Natural Science Foundation of China (Grant agreement 31872486).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The vast tropical Pacific is home to the majority of tropical cyclones (TCs) which threaten the rim countries every year. The TC genesis is nourished by warm sea surface temperatures (SSTs). During El Niño, the western Pacific warm pool extends eastward. However, the number of TCs does not increase significantly with the expanding warm pool and it remains comparable between El Niño and La Niña. Here, we show that the subsurface heat content change counteracts the favorable SSTs in the tropical central-north Pacific. Due to the anomalous positive wind stress curl, the 26 °C isotherm shoals during El Niño over this region and the heat content diminishes in the tropical central-north Pacific, even though warm SST anomalies prevail. This negative correlation between SST and 26 °C isotherm depth anomalies is opposite to the positive correlation in the tropical eastern and western Pacific. This is critical because quantifying the dynamics of the subsurface ocean provides insight into TC genesis. The trend in TC genesis continues to be debated. Future projections must account for the net effect of the surface-subsurface dynamics on TCs, especially given the expected El Niño-like pattern over the tropical Pacific under global warming. Wind stress curl anomalies break the ocean dynamical bond between sea surface and subsurface, constraining tropical cyclone genesis in the tropical central-north Pacific, leading to comparable number of TC for El Niño and La Niña in the western North Pacific. Due to global warming, sea surface temperatures (SSTs) over the tropical Pacific have warmed significantly3 even though the fate of the east–west gradient in the deep tropics is debated5. It has been proposed that the easterly trade winds tend to weaken due to the reduction of the zonal SST gradient6, favoring an El Niño-like pattern in the tropical Pacific10. However, much debate has occurred over the opposite secular trend during the first decade of the 21st century where the east–west SST gradient and the trade winds have strengthened leading to what is referred to as the “hiatus”11. The “hiatus” was declared to have ended since approximately 2011, resulting in an El Niño-like pattern of warming12. Therefore, an examination on the contrasts in TC genesis between El Niño and La Niña, the two phases of El Niño-Southern Oscillation (ENSO), is an analog to advance our understandings of TC genesis under global warming.The genesis of tropical cyclones (TCs) requires energy from the warm ocean13. Warm (cold) SST anomalies occur in the tropical eastern and central Pacific during El Niño (La Niña), due to a coupling between SSTs, surface winds, and the thermocline17. ENSO can modulate the number of TCs19 by modulating the ocean and atmosphere states. The western Pacific warm pool expands significantly eastward during an El Niño20. However, the total number of TCs generated in the tropical western North Pacific is not significantly different between El Niño and La Niña  has been proposed for the western North Pacific TCs30, i.e.,31; F is the net longwave radiation at the sea surface; p is a coefficient which enables the best fit of GPIocean to observations. The dependence of TC numbers and −1 over most of the northwestern Pacific Ocean32 and thus generally not large enough to prohibit TC genesis. Indeed, vertical wind shear was tested but GPIocean was not found to be as sensitive to it as the factors listed above and was not retained in the GPIocean calculation. The suitability of GPIocean over other GPIs formulation for the western North Pacific TCs is that the ocean heat content is explicitly represented in terms of the depth of 26 °C isotherm, which facilitates the quantitative analyses in our study.The thermodynamics of TCs is ideally modeled as a Carnot heat engine, running between a warm reservoir (the ocean) and a cold reservoir (the troposphere)ocean are shown in Fig. ocean show that more than 90% of TCs originate to the west of 160°E during La Niña, while TC genesis locations extend eastward to about 170°E during El Niño albeit with a similar number of total TCs over the tropical western North Pacific. The performance of GPIocean is consistent with GPIs defined by (28) and (29), as shown in Supplementary Fig. ocean (The TC genesis numbers during the peak typhoon season (from July to October) estimated with GPIocean on each variable , it has been well documented and understood that ocean  generated per month in the tropical central-north Pacific  and the SST changes must be considered in total for TC genesis in a warming world. ENSO impact on TC genesis provides an excellent analog and a cautionary tale for a potential cancellation of the dynamic and thermodynamic impacts on GPI.It should be emphasized that there is no evidence of a difference in these processes due to different ENSO types41 after being born over the tropical central Pacific. However, the importance of the need to consider the combined effects of SST and ocean heat content on TC genesis can hardly be overemphasized, especially in a warming world.A caveat here is that there are uncertainties which are expected to be eliminated by further analysis. For example, the main results have a quantitative dependence on the form of GPI . In this study, a TC is generated when the maximum sustained wind speed reaches 34 knots for the first time. The TCs generated over the North Pacific which travel westward and threaten eastern Asia are considered in this study.42 developed by the National Centers for Environmental Prediction (NCEP). The horizontal resolution is 1/3° latitude × 1° longitude. The Simple Ocean Data Assimilation (SODA) version 3 reanalysis products43 and the monthly Extended Reconstructed SST (ERSST) version 5 dataset44 are also used. The results using SODA reanalysis and ERSST are qualitatively the same as the ones using GODAS. In addition, observations from Argo, i.e., BOA_Argo45 are used to verify the results obtained from the analysis and reanalysis products.For the ocean variables, monthly ocean temperatures are obtained from the Global Ocean Data Assimilation System (GODAS) data products46 with a resolution of 2.5° latitude × 2.5° longitude. The NCEP-DOE Reanalysis II products47 and ERA5 reanalysis48 products at European Centre for Medium-Range Weather Forecasts (ECMWF) are also applied.For the atmosphere variables, wind velocities, specific humidity, air temperatures and surface heat fluxes are obtained from the monthly National Center for Environmental Prediction-National Center for Atmospheric Research (NCEP/NCAR) Reanalysis I products49 (ONI) is used to define El Niño and La Niña. It is calculated as monthly SST anomalies in the Niño 3.4 region (5°S–5°N and 120°W-170°W) after a 3-month running mean. When the monthly ONI is higher (lower) than 0.5 °C (−0.5 °C) for at least 5 consecutive months, an El Niño (La Niña) event is defined. The peak typhoon season is from July to October. All months in the peak season that fall into the El Niño and La Niña events are listed in Table The Oceanic Niño IndexBased on the definition of El Niño, when the largest SST anomaly occurs to the east (west) of 150°W, the El Niño event is defined as an EP (CP) El Niño. The months for the CP- and EP-type El Niño during the peak season is classified in Supplementary Table 53. Although the GPI is empirical and not based on the dynamical and physical constraints, it captures the intrinsic dynamical constraints during TCs and often out-performs the dynamical approaches56. The conclusions in this study are not sensitive to the specific form of GPIocean. Particularly, another GPIatm_ocean was proposed in Eq. atm_ocean, which is qualitatively consistent with Fig. ocean and GPIatm_ocean are reproduced with HighResMIP outputs and the same conclusion can be drawn from them as well (Supplementary Fig. GPI is a statistical proxy for the TC dynamics in nature. It has been widely used to quantify the influences of various physical drivers on TCsThe Ekman pumping and suction is calculated asSupplementary InformationPeer Review File


Here, we describe the inverse mechanism for activation of protein kinase D (PKD), an essential kinase involved in membrane trafficking. We show that inactive PKD is dimeric and that its activation depends on dissociation of its kinase domains followed by activation loop autophosphorylation in cis (by itself). Nature has, therefore, found two solutions to the same problem that are simply the inverse of one another.Many eukaryotic protein kinases are activated by phosphorylation of their activation loop. Some protein kinases can autoactivate by phosphorylating their own activation loop. This reaction is widely believed to occur exclusively in  trans, whereby transient dimerization leads to the mutual phosphorylation of the activation loop of the opposing protomer. In this study, we demonstrate that protein kinase D (PKD) is regulated by the inverse mechanism of dimerization-mediated trans-autoinhibition, followed by activation loop autophosphorylation in cis. We show that PKD forms a stable face-to-face homodimer that is incapable of either autophosphorylation or substrate phosphorylation. Dissociation of this trans-autoinhibited dimer results in activation loop autophosphorylation, which occurs exclusively in cis. Phosphorylation serves to increase PKD activity and prevent trans-autoinhibition, thereby switching PKD on. Our findings not only reveal the mechanism of PKD regulation but also have profound implications for the regulation of many other eukaryotic kinases.Phosphorylation is a ubiquitous mechanism by which signals are transduced in cells. Protein kinases, enzymes that catalyze the phosphotransfer reaction are, themselves, often regulated by phosphorylation. Paradoxically, however, a substantial fraction of more than 500 human protein kinases are capable of catalyzing their own activation loop phosphorylation. Commonly, these kinases perform this autophosphorylation reaction in  Protein phosphorylation is the most abundant and important posttranslational modification in cellular signal transduction . Hence, A major determinant for the activity status of a protein kinase is the trajectory of a flexible loop within the kinase called the activation loop. While its inactive conformation can vary greatly , active cis and trans , where its activity is required for the formation of secretory cargo carriers of the TGN to the cell surface (CARTS) (One example of a kinase reported to autophosphorylate is protein kinase D (PKD). PKD is a family of Ser/Thr kinases of the calcium/calmodulin-dependent kinase (CAMK) family comprising three isoforms, PKD1, 2, and 3, which all share the same domain arrangement A. In epi (CARTS) , 11. PKD (CARTS) , 13, whi (CARTS) , 15. A s (CARTS) , 16, alt (CARTS)  among ot (CARTS) , 19. In trans but in cis. Finally, we show that dissociation of the kinase domains of inactive PKD dimers and their subsequent activation loop autophosphorylation is essential for constitutive secretion in cells.In this study, we biochemically dissect the mechanisms of autoinhibition and activation of PKD. We show that the kinase domain of PKD, counterintuitively, forms an inactive dimer. We demonstrate that activation loop autophosphorylation is promoted by dissociation of the inactive dimer and that phosphorylation both increases kinase activity and prevents reassociation of the kinase domain. We present biochemical evidence that autophosphorylation and substrate phosphorylation are mechanistically distinct and that activation loop autophosphorylation occurs, not in PKD activation and autophosphorylation in cells are dependent on the formation of a homodimer that is mediated via its N-terminal ULD and C-terminal kinase domain. The unphosphorylated kinase domain of PKD forms a stable dimer in solution, while activation loop phosphorylation prevents its dimerization , but theSI Appendix, Figs. S1 and S2). The PKD1 kinase domain construct, henceforth referred to as PKD1KD, comprises residues 570 to 890 and includes, in addition to the canonical kinase domain, short flanking regions with high sequence conservation in vertebrate PKD orthologs. The oligomeric state of each protein was evaluated by analytical size exclusion chromatography .We first used the comparative modeling tool of the Rosetta in silico modeling suite  to predi binding E. Compar binding  shows stKD , cross-linking was performed under conditions of saturating ATP and MgCl2. Mass spectrometric analysis of cross-linked residues in both the monomeric and the dimeric species isolated from the gel . Cross-links from the activation loop residues K737 to E668 in the αD-helix and E624 in the β3-αC loop were unique to the monomer, while cross-linking of E736 in the activation loop and K622 in the β3-αC loop was reduced in the dimer. Together with an abundance of monomer-specific loop links that are not observed in the dimer, this indicates a pronounced loss of activation loop flexibility in the dimer. The highly abundant loop link (K622–E624) within the β3-αC loop of the monomer disappeared completely in the dimer and was replaced by a high-confidence intermolecular cross-link between K622 in the β3-αC loop and E668 in the αD-helix. This observation is compatible with a face-to-face arrangement of the PKD kinase domain in the dimer, which has previously been observed experimentally for Chk2, the most closely related kinase to PKD for which a dimeric structure has been determined  that forms an intricate network of salt bridges and hydrogen bonds with R741 and S742 in the activation loop and N757 in the αE/F-αF loop . Activation loop phosphorylation on S742 or the mutagenesis of S738 and S742 to glutamate (PKDKD 2E) also prevents dimerization carbodiimide hydrochloride (EDC) on dephosphorylated PKD1KD D. Since  the gel D revealetermined . The res-αF loop G. This irization C and H . Furthermore, dilution of substoichiometrically phosphorylated PKD1KD resulted in a subsequent increase in autophosphorylation , indicating that the inactive protein is not aggregated. We therefore concluded that the kinase domains must dimerize in a manner that inhibits their ability to autophosphorylate. The affinity of homodimerization of the kinase domain on the basis of trans-autoinhibition was estimated to be 0.5 to 1.4 μM. To assess whether dimerization also affects substrate phosphorylation, we fused the homodimeric ULD of PKD1 . Both mutants showed comparable substrate phosphorylation kinetics to wild-type PKD1 . Autophosphorylation at 50 nM was unaffected by the disruption of the interface  with S738E and S742E substitutions in the activation loop (PKD1KD D706N/2E). This protein is dimerization deficient , and cannot be phosphorylated. It is therefore an ideal mimic of PKD1KD in both size and surface properties, while being unable to participate in the reaction. In contrast to both wild-type PKD1KD and PKD1KD E787K, increasing concentrations of PKD1KD D706N/2E did not inhibit autophosphorylation of either PKD1KD or PKD1KD E878K . These observations indicate that molecular crowding does not influence autophosphorylation but rather that an unphosphorylated activation loop is required for trans-autoinhibition.To further test whether molecular crowding could inhibit autophosphorylation by impeding productive collisions between PKD1eficient B, catalyWT and PKD1E787K. As expected, we observed an approximately twofold higher level of activation loop phosphorylation in unstimulated cells for the dimerization mutant, consistent with the inhibitory nature of PKD1 kinase domain dimerization .Finally, we predicted that our dimer-disrupting mutant would lead to enhanced activation loop autophosphorylation in cells. To test this hypothesis directly, we transfected HeLa cells with expression constructs for full-length PKD1cis is highly controversial, and little data exist to support its possibility. Nevertheless, our observations prompted us to ask whether indeed it could be the mechanism by which PKD activates itself. A cis-reaction should be both concentration independent and limited by the intrinsic enzymatic rate of phosphotransfer, which should result in linear kinetics until the reaction reaches stoichiometric completion. To test whether this is the case for PKD1, we analyzed the kinetics of autophosphorylation over a range of PKD1KD concentrations at which the kinase domain is not trans-autoinhibited (10 to 50 nM). The kinetics of phosphate incorporation were well approximated by a linear model at each concentration before reaching a plateau . Incubation of 10 nM wild-type PKD1KD with an excess of 40 nM PKD1KD D706N resulted in the incorporation of phosphate to a level that corresponds to 10 nM wild-type PKD1KD and not to 50 nM total PKD1  with PKD1KD D706N at a ratio of 1:4 and a total concentration of 50 nM. Mass spectrometry of the resulting mixture revealed the incorporation of up to four phosphates exclusively into wild-type PKD1KD but not kinase-inactive PKD1KD .The observation that the artificial dimerization of the PKD kinase domain by fusion to a dimerization domain accelerates autophosphorylation kinetics previously led us to propose PKD autophosphorylation in KD 0P to PKD1KD 2E in the presence of both ATP and Mg2+. In PKD1KD 2E compared to PKD1KD 0P, we observed a strong and very specific decrease in the rates of HD exchange in the activation loop and the catalytic loop as well as the αC-helix and β1 and β3 strands . Together, these elements constitute the kinase active site, and their stabilization is consistent with the stereotypical role of activation loop phosphorylation. Correspondingly, the catalytic efficiencies of stoichiometrically S742-phosphorylated PKD1KD (1P) and PKD1KD 2E were 9- and 17-fold higher, respectively, compared to those of unphosphorylated PKD1KD (0P)  C.SI Appendix, Fig. S7B), which excludes it from the classical group of RD kinases. While it has been postulated that only RD kinases can be regulated by activation loop phosphorylation . Mutation of R732 to methionine in PKD1KD abrogated the increase in catk gained by phosphorylation or phosphomimetic substitutions  , consistent with a defect in coordination of the phosphorylated activation loop and not a defect in autophosphorylation.Typically, kinases that depend on activation loop phosphorylation to acquire full activity rely on a conserved arginine in the so-called HRD motif to make electrostatic interactions with the respective phosphate group and thereby stabilize the activation loop in its active conformation. PKD, however, encodes a cysteine at this position  , lead to activation loop hyperphosphorylation in cells  that is not exhibited by either the phosphorylation motifs (S738 and S742) in the activation loop or an additional autophosphorylation motif in the C terminus of PKD1 and PKD2 A. Kinasein vitro B, and th GPKDrep  was signin cells C. In vite PKD1KD D. We nexactivity . InteresA and B)  15). Mu. MuKD D6in cells D and 5 EK612W , while PAUF secretion was completely abrogated  is dimeric in vitro , despite the fact that unphosphorylated PKD1KD (0P) has substantial basal activity in vitro . This is presumably due to dissociation of the kinase domain dimer upon low, but not absent, activation loop phosphorylation in stimulated cells . Disruption of kinase domain dimerization either by introduction of phosphomimetics in the activation loop (2E) or by mutation of the αG interface (E787K) resulted in membrane translocation kinetics that were indistinguishable from PKD1WT . Finally, we combined a mutation in the ULD dimer interface (F104E in PKD1 and F59E in CeDKF1) (F104E+S738E/S742E) to create a construct which is compromised in all known dimerization interfaces. This construct, which is presumably monomeric, also did not show altered membrane translocation kinetics . We therefore conclude that the autoregulation of DAG binding does not intrinsically depend on PKD dimerization.Previous work has established that the DAG-binding C1 domains of PKD are sequestered in its autoinhibited, cytosolic conformation . The end CeDKF1) C 15) wi wiSI AppSI Appendix, Fig. S12E), mutation of the catalytic aspartate in the active site to asparagine (D706N) had no effect on membrane binding . These observations indicate that ATP binding, but not catalysis, is important for stabilization of the intramolecular, autoinhibited assembly. Interestingly, PKD1D665N, which exhibited a similar enrichment at the TGN, also translocated to the plasma membrane significantly faster than wild-type PKD1 . In addition to mediating substrate binding, D665 is predicted to make hydrogen bonds to the ribose moiety of ATP, suggesting that nucleotide binding is critical for stable PKD autoinhibition.Previous studies have shown an enrichment of kinase-dead PKD at the TGN when compared to that of wild-type PKD . Intrigutrans. This necessitates their transient dimerization. In this study, we demonstrate that kinases can be regulated by the inverse mechanism: dissociation of an inhibitory dimer followed by autophosphorylation in cis  was performed in 40 mM MES pH 6.5 and 100 mM NaCl. 4 µM protein containing 1 mM ATP and 2 mM MgCl2 was mixed with varying concentrations of EDC and Sulfo-NHS at a constant ratio of 1:2.5 and incubated for 30 min in the dark. The reaction was quenched with 20 mM β-mercaptoethanol and 50 mM Tris pH 7.5. Cross-linked monomeric and dimeric species were separated by SDS–PAGE. Details of the XL–MS sample analysis can be found in SI Appendix.Cross-linking of PKD1Homology modeling of the PKD1 kinase domain (residues 576 to 873) was performed using the Rosetta Comparative Modeling tool  and the Homology modeling of the PKD1 kinase domain dimer was performed using the Rosetta Comparative Modeling tool and symmetry imposed by the structure of Chk2 (PDB 3i6u). Additional constraints were imposed based on dimer-specific intramolecular EDC cross-links (615 to 622) and intermolecular EDC cross-links (622 to 831) and an EDC cross-link common to both monomer and dimer (616 to 624), with a distance constraint of 3.8 Å. The model was tested biochemically by introducing a single point mutation (E787K) into the predicted dimer interface.KD at 2 mg/mL was injected onto an S200 10/300 column (Cytiva) connected to a 1260 Infinity HPLC (Agilent Technologies). A miniDAWN TREOS (Wyatt) was used to detect light scattering at 690 nm, and a Shodex RI-101 (Shodex) detector was used for refractive index measurement. All runs were performed at room temperature in 20 mM HEPES pH 7.4, 150 mM KCl, 1 mM EDTA, 1 mM TCEP, and 1% (v/v) glycerol.For determination of particle mass and polydispersity, 60 µL of purified PKD12, and 1 µL [γ-32P] ATP/100 µL reaction and were terminated by the addition of 10 mM EDTA. The radioactivity from phosphorylated material immobilized in polyacrylamide gels or on 0.45-μm nitrocellulose membranes (Cytiva) was detected by exposure to a phosphor screen, imaged by an Amersham Typhoon phosphorimager, quantified in ImageJ, and converted into molar quantities by calibration with internal standards. Details of the different kinase assays performed can be found in SI Appendix.All kinase assays were done with radiolabeled [γ-32P] ATP  in a reaction buffer containing 50 mM HEPES pH 7.4, 150 mM KCl, 1 mM TCEP, 1 mM EDTA, 1% (v/v) glycerol, 0.05% (v/v) Tween-20, 1 mM ATP, 5 mM MgClKD and PKD1KD S738/742E were conducted in a final reaction volume of 6 µL with a final concentration of 5 µM (30 pmol) PKD1KD or PKD1KD S738/742E. The reaction was initiated by the addition of 5.25 µL of D2O buffer ) to 0.75 µL of PKD1cat WT or mutant . The reaction proceeded for 3, 30, 300, or 3,000 s at 20 °C before being quenched with ice-cold acidic quench buffer, resulting in a final concentration of 0.6 M guanidine HCl and 0.9% formic acid postquench. All conditions and time points were generated in triplicate. Samples were flash frozen immediately after quenching and stored at −80 °C until injected onto the ultraperformance liquid chromatography (UPLC) system for proteolytic cleavage, peptide separation, and injection onto a QTOF for mass analysis, described below. Details of the HDX–MS analysis can be found in SI Appendix.HDX reactions for wild-type PKD1mycoplasma contamination using MycoAlert . For transient plasmid transfections, HEK293T cells were transfected with TransIT-293 (Mirus Bio). HeLa cells were transfected with TransIT-HeLaMONSTER (Mirus Bio).HeLa and HEK293T cells were maintained in RPMI 1640 medium supplemented with 10% FCS. Cell lines were authenticated using Multiplex Cell Authentication by Multiplexion  as described recently . The SNP profiles matched known profiles or were unique. Cells were tested negative for SI Appendix.Flp-In T-REx-HeLa cells  were grown in DMEM containing 10% FCS, 100 µg/mL zeocin, and 10 µg/mL blasticidin (HeLa). These cells stably express the Tet repressor, contain a single Flp Recombination Target (FRT) site, and were used to generate the Flp-In-T-REx-PKD1-GFP lines. Cells were cotransfected with pcDNA5/FRT/TO-PKD1-GFP and the Flp recombinase expression plasmid pOG44 at a ratio of 1:10 and then selected with 500 µg/mL hygromycin B. Induction of protein expression with doxycycline was at 10 ng/mL. Details of the immunofluorescence staining and confocal microscopy can be found in FlpIN Trex HeLa cells stably expressing PKD1-EGFP variants WT, K612W, and D655N were transfected with a plasmid encoding PAUF-Myc-His, and after another 24 h, the expression of PKD1-EGFP was induced with doxycycline. HEK293T cells were transiently transfected with plasmids encoding PKD1-EGFP variants WT, S2E, S2A, R732M, or R732M S2E and PAUF-Myc-His in a ratio of 1:5. Growth medium was removed 24 h posttransfection, and cells were incubated in serum-free medium. After 5 h, the medium was collected, and cells were lysed. Aliquots of medium and detergent-soluble cell proteins were assayed for PAUF content by western blot analysis.Appendix 01 (PDF)Click here for additional data file.


One of those is the 4(1H)-quinolone series which inhibits cytochrome 1bc and effectively kills drug-resistant malaria parasites. However, some of these compounds have unexpected toxicity due to cross-species inhibition of human cytochrome 1bc. In this work, we explore by experimental and computational studies how 4(1H)-quinolone compounds work with human and parasite cytochrome 1bc. This information reveals the key to improved selectivity between human and parasite cytochrome 1bc and helps drug developers to design new compounds with better therapeutic efficiency and safety.Malaria is a life-threatening disease which infects millions of people a year via mosquito bites, particularly in developing countries. Although many malaria drugs are available in the market today, all of them have been challenged by drug-resistant variants. Developing a new drug to fight mutated malaria is extremely critical. Cytochrome o) site of the Plasmodium falciparum bc1 complex, such as atovaquone, have become less effective due to the rapid emergence of resistance linked to point mutations in the Qo site. Recent findings showed a series of 2-aryl quinolones mediate inhibitions of this complex by binding to the ubiquinone-reduction (Qi) site, which offers a potential advantage in circumventing drug resistance. Since it is essential to understand how 2-aryl quinolone lead compounds bind within the Qi site, here we describe the co-crystallization and structure elucidation of the bovine cytochrome 1bc complex with three different antimalarial 4(1H)-quinolone sub-types, including two 2-aryl quinolone derivatives and a 3-aryl quinolone analogue for comparison. Currently, no structural information is available for Plasmodial cytochrome 1bc. Our crystallographic studies have enabled comparison of an in-silico homology docking model of P. falciparum with the mammalian’s equivalent, enabling an examination of how binding compares for the 2- versus 3-aryl analogues. Based on crystallographic and computational modeling, key differences in human and P. falciparum Qi sites have been mapped that provide new insights that can be exploited for the development of next-generation antimalarials with greater selective inhibitory activity against the parasite 1bc with improved antimalarial properties.Antimalarials targeting the ubiquinol-oxidation (Q Malaria remains a devastating disease: it was estimated that in 2020 there were ~241 million cases and 627,000 deaths, primarily in children under 5 years old. Although much progress has been made to reduce the rate of malaria mortality and morbidity .1bc is a multi-subunit protein complex embedded in the inner mitochondrial membrane [b, cytochrome 1c, and the Rieske iron-sulfur protein, which are responsible for proton translocation through the membrane and electron transfer to cytochrome c via the redox reaction of ubiquinone in the Q cycle [b subunit; the ubiquinol-oxidation (Qo) site and the ubiquinone-reduction (Qi) site [1bc has a critical function to re-generate ubiquinone for dihydroorotate dehydrogenase (DHODH), which is an important enzyme in parasites’ de novo pyrimidine biosynthesis pathway [Toxoplasma gondii, Trypanosoma cruzi, and Leishmania donovani [TM) is a hydroxynaphthoquinone and is widely-used in the treatment and prevention of uncomplicated malaria [Plasmodium spp.) and toxoplasmosis (Toxoplasma gondii) [o site of cytochrome 1bc and ultimately disrupting de novo pyrimidine biosynthesis [o site, especially Y268S, weaken the binding of atovaquone to the Qo pocket, leading to the emergence of atovaquone-resistant malaria strains [The mitochondrial electron transport chain of the malaria parasite is a validated target for antimalarial drug development . Cytochrmembrane  as part  Q cycle . The Q cQi) site . In para pathway ,9,10. Thdonovani ,12. Atov malaria ,14,15. A gondii)  parasiteynthesis . Point-m strains ,19.1bc from various species have been determined by X-ray crystallography [o site [i site from bovine cytochrome 1bc, a human surrogate with 80% identity with cytochrome b [1bc complex at the Qi site and may be the cause of toxicity. However, a direct link of such a binding to the toxicities observed in preclinical studies needs further investigation. Nevertheless, selectivity between the host and plasmodial targets is a key element in safe-drug design, so understanding differences between the mammalian and parasite Qi targets is a key objective for rational drug design.Structures of cytochrome lography ,24,25,26lography ,28,29. I [o site ,31. In 2chrome b , was crychrome b . Both 4 of P. falciparum, including atovaquone-resistant strains [Chemical compounds based on the 4(1H)-quinolone template have been developed as antimalarials for three quarters of the century . The desbc1 . The disbc1   (o/Qi site of 4(1H)-quinolone-based compounds was determined by monitoring cytochrome 1bc activity in Qo/Qi site in yeast mutants [P. falciparum [o or Qi binding preference. The binding of SCR0911 to bovine cytochrome 1bc was visualized by X-ray crystallography and cryo-electron microscopy [i binder in crystal and in-solution studies. In parallel, in vitro assessments of 4(1H)-quinolone derivatives, such as CK-2-67, RKA066, and WDH-1U-4  [PfNDH2 is a single-subunit enzyme in the mitochondrial electron transport chain of P. falciparum that acts as an alternative enzyme for typical complex I [1bc [Pf cytochrome 1bc [PfNDH2 [Recently, 2-pyridyl substituted 4(1H)-quinolone SCR0911 A was repression) . The spe mutants ,43 and Plciparum ,45. Subtcroscopy , revealiWDH-1U-4 B, were r(PfNDH2) ,46 -quinolones, CK-2-67, RKA066, and WDH-1U-4, to investigate the binding modes of these inhibitors within the Qi site of representatives of 2-aryl and 3-aryl quinolone. Due to the lack of 1bc structure from P. falciparum, we generated the homology model of Pf cytochrome 1bc and explored possible inhibitor binding modes of these antimalarial compounds by using molecular docking.We co-crystallized bovine cytochrome 1bc was purified from mitochondria, as previously described [1bc fractions were pooled and concentrated in a centrifugal ultrafilter (MWCO. 100 kDa) and loaded onto a 120 mL Sephacryl S300 column  equilibrated in and eluted at a flow rate of 0.5 mL/min with 20 mM KMOPS pH 7.2, 100 mM NaCl, 0.5 mM EDTA, 0.01% DDM. Cytochrome 1bc fractions were pooled and concentrated to 40 mg/mL. PEG fractionation with an increasing amount of PEG4000 was used to precipitate cytochrome 1bc. The protein started precipitating at 2% PEG4000, and pure protein was in the fraction of 2.5–4% PEG4000. Cytochrome 1bc pellet was re-solubilized in 25 mM KPi pH 7.5, 100 mM NaCl, 0.5 mM EDTA, and 0.015% DDM and dialyzed in the same buffer in a centrifugal ultrafilter to remove residual PEG. The inhibitors were dissolved in DMSO at the concentration of 50 mM and 5 μM cytochrome 1bc was incubated with 50 μM inhibitor (10-fold molar excess) at 4 °C for 12 h.Cytochrome escribed . Crude mescribed  and solu1bc was mixed with 1.6% HECAMEG and adjusted to the protein concentration of 40 mg/mL. In total 2 μM of final protein solution was mixed with 2 μM of reservoir solution . The crystals were grown using the hanging drop method at 4 °C to the mature size of ~100 μm within four days. The single crystals were soaked stepwise in 50% ethylene glycol with reservoir solution prior to freezing in liquid nitrogen. Two datasets 1bc-CK-2-67 and 1bc-RKA066 were collected at PROXIMA-1 beamline  at 100 K, using 0.9786 Å wavelength on EIGER-X 16 M detector and 0.9786 Å wavelength on Pilatus3 6M, respectively, 1bc-WDH-1U-4 data were collected at PROXIMA-2A beamline with 0.9800 Å wavelength using EIGER X 9M detector . All data were processed by iMosflm [1bc (PDB:5OKD) without inhibitors or ligands as the starting model. The model was manually rebuilt in COOT [The inhibitor-bound cytochrome  iMosflm . The str iMosflm  using bo in COOT  between  in COOT . Data coP. falciparum 3D7 parasites were cultured in human erythrocytes  at 5% haematocrit under 5% CO2, 2% O2, and 93% N2. Cultures were grown in complete medium (CM) containing RPMI 1640 medium (Gibco) supplemented with 25 mM HEPES (VWR), 50 μg/mL hypoxanthine, 0.25 mM NaHCO3, 50 μg/mL gentamicin sulfate, and 10% pooled heat inactivated AB + human serum [50 values were calculated from the log of the dose/response relationship, as fitted with Grafit software version 7.0.3 .an serum . Parasitan serum . Drug suan serum . Drug ICCK-2-67, RKA066 and WDH-1U-4 were synthesized as described by Pidathala C. et al. .1H NMR, as follows, which matched previous report values.The structures of these three compounds were confirmed by 1H NMR  δH 8.98 , 8.27 , 7.60 , 7.56 , 7.9 , 7.26 , 7.20 , 7.16 , 7.11 , 3.96 , 2.01 ;CK-2-67: 1H NMR  δ 11.20 , 8.23 , 7.84 , 7.73 , 7.46 , 7.44 , 7.39 , 7.35 , 7.32 , 4.07 ,1.75 .RKA066: 1H NMR  δ 11.60 , 8.05 , 7.61 , 7.51 , 7.44–7.38 , 7.33–7.21 , 7.16 , 4.00 , 2.20 .WDH-1U-4: All three compounds showed >95% purity when tested by the HPLC method described here. HPLC analysis was carried out using the Agilent 1200 system. The HPLC method used the following conditions: ZORBAX Eclipse Plus C18  at 25 °C with 1.0 mL/min flow rate; solvents: (A) water containing 0.05% trifluoroacetic acid and (B) acetonitrile containing 0.05% trifluoroacetic acid; method : run time: 15 min, gradient: 2% B hold to 2 min, 2–98% B in 10 min, then hold at 98% B to 15 min.P. falciparum is retrieved from UniProtKB (ID: Q02768). Pf cytochrome b homology model was generated using web-based protein modelling server: SwissModel software  [b template (chain c of 5OKD model). The model was built based on target-template alignment using ProMod3 version 1.1.0  [Pf homology model used in this paper has a QMEAN score of −5.67 and 41.71% sequence identity.The primary sequence of zerland) ,59,60 wizerland) . The conzerland) . The Pf Pf Qi site, using the “Accurate” parameter, allowing flexibility for the sidechain within 5 Å to its reference binding mode. The final solutions were determined based on binding pose corresponding to the solved bovine crystal structure with the highest Fullfitness score  [Full details of molecular docking to the P. falciparum homology model are provided in ss score . All nonstralia) .1bc inhibition assays were measured in triplicate in 50 mM KPi pH 7.5, 2 mM EDTA, 10 mM KCN, 30 μM equine cytochrome c , and 2.5 nM bovine cytochrome 1bc at room temperature. The inhibitors were added to the assay without prior incubation. The reaction was initiated by the addition of 50 μM decylubiquinol . The reduction of cytochrome c was monitored by the different absorption between 550 and 542 nm using extinction coefficient of 18.1 μM−1cm−1 in a SPECTRAmax Plus 384 UV-visible Spectrometer .Bovine cytochrome P. falciparum (TM90C2B) were reported in previous studies and are summarized in bc1 represents a potential for increased mammalian/human safety. The bovine 1bc inhibition level of studied 4(1H)-quinolones and 4(1H)-pyridones were measured in this work. Amongst all the compounds tested, CK-2-67 and RKA066 has the strongest bovine 1bc inhibition at 0.1 µM  compared to WDH-1U-4 (7%). Meanwhile, the pyridone GSK932121 showed a 65% inhibition in bovine bc1 at 0.1 µM, suggesting that the presence of the NH and N-OH group within the core may enhance mammalian 1bc inhibition, leading to poor selectivity -quinolones and 4(1H)-pyridones against drug-sensitive (3D7) and atovaquone-resistant ectivity .bc1 co-crystallized with CK-2-67, RKA066, and WDH-1U-4 in the resolution range of 3.3–3.5 Å -quinolone analogues extends away from head group into hydrophobic cavity between Phe18, Gly38, Ile39, and Ile42 residues, and the trifluoromethoxy group is positioned between Met190 and Met194.The 4(1H)-quinolone ring orientation in the 3-aryl-4(1H)-quinolone (WDH-1U-4) is similar to the orientation of the 4(1H)-pyridone ring in GSK932121 but lies in a reversed direction to the other 2-aryl-4(1H)-quinolones studied c,e,g . The more flexible sidechain, 2-aryl-4(1H)-quinolones, of CK-2-67 and RKA066 has the carbonyl group closer to His201, and the NH group is closer to Asp228 . The sequence of Pf cytochrome b has a number of unique features from human sequences that may enable drug selectivity -quinolone core into the apex of Qractions . The N-hh His192 a.1Pf bc, which is consistent with the molecular docking results, which showed only hydrophobic interactions and the lack of hydrogen bonds or aromatic stacking. In contrast, CK-2-67 and GSK932121 can form crucial hydrogen bonds between the hydroxyl methyl group and His192 displayed higher potency in both phenotypic and enzymatic 1Pf bc assays than CK-2-67 -quinolone cores of ELQ300 and SCR0911 are located close to His192 in this in silico analysis.The 4(1H)-quinolone core of WDH-1U-4 was predicted to be at a hydrogen bonding distance to the main chain oxygen of His12 with the trifluoromethoxy phenyl sidechain of this compound forming favorable interactions with Tyr16, Phe30, Ile34, Phe37, and Phe210 d. This lcompound . Notablyhan 1 nM , and it  Qi site e,f. The  Qi site e, and th Qi site f. These Pf parasite growth compared to the 4(1H)-quinolones with phenoxy-phenyl (ELQ300) and phenyl-pyridyl sidechain (SCR0911) (Pf parasites with Qo site mutations (TM90C2B), where CK-2-67 and RKA066 have low potency with triple-digit nanomolar IC50, while ELQ300, SCR0911, and WDH-1U-4 retained their potency.The 4(1H)-quinolones with bis-aryl tail  displayed weaker inhibition of SCR0911) . This tro/Qi site selectivity, the availability of either an inhibitor-bound 1Pf bc structure or P. falciparum assay of Qo/Qi mutated strains could be beneficial. However, in the absence of those, this computational study brought us to the conclusion that the flexibility of aromatic side chain could be a factor affecting the binding affinity to Pf Qi site. It is clear that the rigid phenyl-pyridyl sidechain of SCR0911 forms strong π-π stacking with both Phe30 and Phe37 , such as the methoxy group, could be added to assist hydrogen bond formation with Tyr16. As incorporation of 3-ester in a similar series of 4(1H)-quinolones always delivered potent antimalarial activity [2) in our series could be replaced by a larger alkyl group, such as ethyl or iso-propyl, which could fill the space near the Leu188-like 3-ester group with more hydrophobic interactions. In order to minimize toxicity in the host (1) should minimize electrostatic interaction between 4(1H)-quinolone head and Ser35 of human 1bc. RKA066 (R1: OH) is an example, where R1 tightly binds to Asp218 of 1Pfbc but has cross-reactivity to human cytochrome bc1 due to hydrogen bonds with Ser35. However, N-methyl quinolone was reported to be less effective than its counterpart to inhibit P. falciparum [1 to develop new compounds. Strong intermolecular interactions between 4(1H)-quinolone rings can cause poor aqueous solubility, which hampers the development of new compounds. To increase solubility and pharmacological success of the new compounds, ring A or ring C could be replaced by saturated heterocycles, including a degree of polarity and reducing flatness of the molecule [Following the determination of co-crystallographic bovine  Qi site . Interaclciparum a,b are eactivity , the metthe host c,d, growlciparum . Carefulmolecule .1bc from bovine and humans have high sequence conservation for both Qo and Qi sites: 94% and 80%, respectively. The superimposed crystal structures of bovine [1bc [o and Qi sites that interact with inhibitors are well-conserved between both species -quinolones inhibitors validated their position within the bovine Qd, JAG21 . This flsolution . This fli pocket, which ubiquinone binds before reducing to ubiquinol within the Q cycle [i inhibitors, i.e., antimycin [i pocket and ultimately disrupt the Q cycle. The additional hydrogen bonds between the quinolone core to His201 and Ser35 make CK-2-67 and RKA066 a more potent inhibitor of bovine 1bc. Moreover, the presence of NH and N-OH in the core of CK-2-67 and RKA066 might enhance the inhibition of mammalian 1bc, leading to poor selectivity. For the pyridone GSK932121, there is an additional hydrogen bond between its hydroxyl-methyl group and His201. This additional H-bonding likely plays a role in the strong inhibitory effects seen versus the bovine 1bc complex of 65% at 0.1 µM. The results confirm that including the N-hydroxy and methoxy sidechains increases drug solubility. The poor physicochemical properties associated with these molecules may lead to greater off-target toxicity, thereby reducing the potential therapeutic index.His201 and Asp228 are key amino acid residues in the Qntimycin ,69, NQNOntimycin , and ascntimycin , form hy1bc and explains how some compounds could bind to the Qi site more strongly than others. The significance of this focused study is that it shows that the 3-aryl quinolone derivatives generally have lower off-target mammalian effects  compared with 2-aryl quinolones substituted with similar aryl sidechains. The outlier to this observation is SCR0911, which exemplifies that a curved 2-substituted sidechain can improve the selectivity and provides a template for further targeted studies for lead optimization.The difference in ligand poses and the number of hydrogen bonds between molecule and protein determines their affinity for bovine cytochrome b (notably the loss of histidine and lysine residues in the parasite), a region of the protein adjacent to the catalytically essential ef helix element of Qo, may also help drive drug selectivity [o site can accommodate a greater frequency of mutations than the Qi site without a deleterious effect on enzyme turnover. This is in part supported by the observation that the propensity for the parasite to develop (point-mutation-based) resistance to Qi inhibitors, e.g., ELQ300, is far lower compared to Qo inhibitors, e.g., atovaquone (the reported resistance frequencies were 1 in  105 parasites for atovaquone [8 parasites for ELQ300) [i site potentially an advantageous target over Qo site for exploring selective inhibitor binding to the P. falciparum enzyme. The targeting of the Qi site is additionally attractive for developing antimalarial against existing atovaquone-resistant parasites.Sequence differences in the C-terminal region of the E-ef loop of cytochrome ectivity ,73. It iovaquone  and less ELQ300) . This fe1bc complex from malarial parasites is not available. Molecular docking of antimalarial artemisinins and 4(1H)-pyridones to their target models, including cytochrome 1bc, can describe structure and activity relationship and suggest rational design for new antimalarial lead compounds [P. falciparum cytochrome 1bc (1Pf bc) was generated by SwissModel online platform [b, which has the highest identity in the primary sequence (42%) among the structures from other organisms, was used as a starting model. However, the docking model with flexible ligand, such as ELQ300, shown in the current study might be less accurate [Currently, the structure of cytochrome ompounds ,76,77. Tplatform ,59,60. Taccurate . It is po site in 1Pf bc, while ELQ300, SCR0911, and WDH-1U-4 are much more selective for the Qi site. In support of this hypothesis, a recent study describing the in vitro selection of CK-2-68, a CK-2-67 derivative and resistant parasites, reported mutations in the Qo site [o and Qi sites opening the possibility of a certain degree of cross resistance with atovaquone [This observation suggests that 2-bisaryl 4(1H)quinolones (CK-2-67 and RKA066) might also target the Q Qo site . We and ovaquone ,70.P. falciparum bc1 complex. Rigidity of the aryl sidechain, particularly the curved phenyl-pyridine sidechain in SCR0911, could further enhance inhibition 1Pfbc via pi-pi stacking interactions of Phe30 and Phe37 and thus represents an excellent scaffold for further optimization.Structural studies of bovine cytochrome bc1 complexed with promising antimalarial 4(1H)-quinolones demonstrate that some 2-aryl and 3-aryl 4(1H)-quinolones are specific binders of the Qi site. The 4(1H)-quinolone core of each of the compounds studied  locates in the hydrophilic region at the apex of Qi pocket, while aryl sidechains extend away through the hydrophobic channel. Surprisingly, substitution of the 2- or 3-position of the 4(1H)-quinolone core leads to similar interactions with the Qi site with inversion of the quinolone head group direction within the hydrophilic apex of the Qi site. Through the detailed analysis of seven molecules covering lead compounds and drug candidates from the quinolone and pyridone families, it is proposed that the number of hydrogen bonds with Qi site residues is the most important factor contributing to strong inhibition of the bovine enzyme, as exemplified by RKA066 and GSK932121. In silico docking revealed that the flexibility of the tail group is an important factor in binding to the P. falciparum provides new opportunities for structure-based lead optimization and drug design. Binding analyses of 4(1H)-quinolones in P. falciparum and human Qi sites generated from molecular docking and bovine crystal structures suggest several interactions that could be used for development of the next generation 4(1H)-quinolone lead compounds with improved selectivity against P. falciparum with reduced toxicity profiles.The structural differences of the ubiquinone-reduction (Qi) site between the human and


The amount of ice versus supercooled water in clouds is important for their radiative properties and role in climate feedbacks. Hence, knowledge of the concentration of ice‐nucleating particles (INPs) is needed. Generally, the concentrations of INPs are found to be very low in remote marine locations allowing cloud water to persist in a supercooled state. We had expected the concentrations of INPs at the North Pole to be very low given the distance from open ocean and terrestrial sources coupled with effective wet scavenging processes. Here we show that during summer 2018 (August and September) high concentrations of biological INPs (active at >−20°C) were sporadically present at the North Pole. In fact, INP concentrations were sometimes as high as those recorded at mid‐latitude locations strongly impacted by highly active biological INPs, in strong contrast to the Southern Ocean. Furthermore, using a balloon borne sampler we demonstrated that INP concentrations were often different at the surface versus higher in the boundary layer where clouds form. Back trajectory analysis suggests strong sources of INPs near the Russian coast, possibly associated with wind‐driven sea spray production, whereas the pack ice, open leads, and the marginal ice zone were not sources of highly active INPs. These findings suggest that primary ice production, and therefore Arctic climate, is sensitive to transport from locations such as the Russian coast that are already experiencing marked climate change. The concentration of ice‐nucleating particles at the North Pole in summer 2018 was amongst the highest anywhere in the worldBiological ice‐nucleating particles were derived from the Russian seas and perhaps associated with wind‐driven sea sprayThe concentration of ice‐nucleating particles at the surface was often different to that higher in the boundary layer where clouds form The prevailing view is that aerosol within the summertime high Arctic boundary layer experiences little effect from long‐range transport  campaign, which took place throughout August and September 2018 on the Swedish icebreaker Oden. Measurements took place while Oden was on route to the North Pole as well as when it was moored to an ice floe in the inner pack ice and drifting passively between 88 and 90°N. Samples were collected for INP analysis at both ship level (in the surface mixed layer) and using a balloon‐borne sampler in the cloud mixed layer. We use backward trajectories alongside other measurements to suggest that the source of the most active INPs reaching the North Pole is outside of the pack ice, and near the Arctic coast of Russia.2To determine the INP concentration spectra relevant for mixed‐phase clouds in the central Arctic, 48 days of sampling were conducted aboard the Swedish icebreaker Oden during Arctic summertime and the early freeze‐up period (August and September). Filter samples were collected and analyzed during the journey toward the North Pole from Svalbard whilst ice breaking, and whilst moored to an ice floe.2.1−1 (standard temperature and pressure). The inlet was mounted on the 4th deck of the ship, 25 m above mean sea level. This type of filter has been used previously for INP sampling and has a low background INP count and high particle recovery rates  were used to collect aerosol by subsampling from a heated whole‐air inlet at a flow rate of 9 L min−1  and collected on four stages . For substrates on which aerosol were collected we used 25 mm diameter filters of pore size 0.05 μm . In addition, we used an after filter that sits after the impactor plate to collect particles smaller than 0.25 μm (47 mm diameter Nuclepore track‐etched membrane polycarbonate filter with a pore size of 5 μm). Impactor 2 had a flow rate of 100 L min−1  and consisted of three stages . In impactor 2 we used 75 mm diameter Nuclepore track‐etched membrane polycarbonate filters of pore size 0.05 μm. INP spectra from the two overlapping stages were combined in the analysis. Also, the total INP spectrum was defined by summing across all size categories . A radi2.2Filters were analyzed for INP content as soon as possible after sampling, usually within 1–12 hr of being removed from the inlet. The filter samples were not frozen before offline INP analysis, due to concerns that this may affect the INP activity. Instead, they were stored at +4°C. Performing the analysis on ship soon after sampling minimized the risk of changes in the INPs on the filter since storage at any temperature is expected to affect the activity of the samples . The background influence on the INP concentration was determined by collating the differential nucleus concentrations for water and handling blanks, and subtracting this from the sample differential nucleus concentrations. The differential concentrations were then summed to produce the cumulative INP spectra , filter samples of DMS were collected and analyzed onboard. Equivalent black carbon (eBC) concentrations were obtained from a multi‐angle absorption photometer . Particle size distribution measurements were made continuously using an aerosol spectrometer  for particles of size 0.15–9.65 μm, and a differential mobility particle sizer (DMPS) with a custom‐built medium Vienna‐type differential mobility analyzer (DMA) with a mixing condensation particle counter  for particles of size 10–921 nm.An ion chromatography system  was used to determine the chemical composition of the samples. Using certain standards, the concentration of chloride, nitrate, sulfate, mesylate, methane sulfonic acid, sodium, ammonium, potassium, magnesium and calcium in the sample were determined from the ion chromatograms. A synthetic sample  was used to estimate the uncertainty, which was up to 3%. More details on the method can be found in Leck and Svensson .2.4There is contradictory evidence that combustion products in a ship's exhaust can influence INP populations, with some studies indicating that there may be a significant ice nucleating activity . The overall relationship with origin is unchanged by the addition of this filter, which indicates that the results were insensitive to precipitation events.In order to define the potential origin of measured INPs, backward trajectories of the air reaching an altitude of 32m at the sampling location were examined. The 10‐day (only 7 days of which are used here) back trajectories were calculated using the Lagrangian analysis tool LAGRANTO  are as high as the highest INP concentration reported in INP rich regions such as the mid‐latitude terrestrial environment , and highlights the variability of INP concentrations at the North Pole throughout August and September of 2018. The first peak in ice‐nucleating activity was observed during a period in which the ship was breaking ice prior to being moored to an ice‐floe . It is reasonable to question whether the very high INP concentrations observed during the ice‐breaking period resulted from the ice‐breaking itself. Ice‐breaking involved frequent backward and forward motions of the ship, hence there is the potential for sampling ship emissions  and aerosol resulting from ice‐breaking and the disruption of the sea surface. See the methodology for a description of the precautions taken to avoid sampling ship stack (or other) emissions. There was a pause in ice‐breaking when a clean air station was established  that coincided as well with high INP concentrations. At this clean air station, the ship was moored facing into the wind, with ship‐based aerosol sources downwind of the aerosol inlets. Hence, we were confident that sampling of ship pollution and ice‐breaking aerosol were eliminated, increasing confidence that these high values in this period were indeed representative of the central Arctic Ocean. In addition, there was also a period of very high ice‐nucleating activity a few days after the ice‐floe station had been established and the ship was pointing into the wind, demonstrating that there were very active INPs that were not related to ice‐breaking or ship emissions. The time series also highlights two distinct regimes: the period up to the 23rd August was characterized by variable but often very high INP concentrations, whereas after this date INP concentrations were typically much lower. We examine the back trajectories associated with these different regimes later in the paper.The time series in Figure ns) of aerosol particles in Figure ns we used the total aerosol surface area derived from the online DMPS and WELAS instruments sampling from the same whole air inlet that we used to collect the filter samples. ns provides a means of comparing the activity of aerosol on a per unit surface area basis and we compare our results to parameterisations for marine aerosol . The sampler (SHARK), was lofted to a defined height using a tethered balloon operated from the sea ice, and aerosol samples were collected into multiple size bins . DuringT[INP]= 0.1 are shown in Figure T[INP]= 0.1  was around −18 to −19°C above the surface mixed layer, whereas it was below −26°C within it. On the 20th August, T[INP]= 0.1 was around −23°C above the surface mixed layer, but −14°C within it. On all three days, scanning microwave radiometer and six hourly radiosonde temperature profiles confirmed that the surface mixed layer was decoupled from the rest of the boundary layer  is also shown in Figure The size‐resolved INP activity (The coarse mode (>2.5 μm diameter) has a relatively short lifetime in the Arctic boundary layer, being removed effectively by wet scavenging processes  dimethyl sulfide (DMS), a product of marine biological activity, particularly in the marginal ice zone , where the ship was close to open water, but was variable whilst in the pack ice . There is also industry, shipping and mining along the Arctic coast of Russia, and the role of gas flaring is thought to be a particularly important source of BC  is 0.65. There are periods when the eBC concentration and T[INP]= 0.1 track one another, but there are other periods where they vary independently. For example, between the 6th and the 19th August eBC and T[INP]= 0.1 are highly correlated. However, on the 20th August the eBC decreases dramatically, but the INP remain elevated. Conversely, later in the campaign, the eBC values are some of the highest we measured, but the ice nucleating activity remains very low. Hence, we concluded that the eBC indicates long range transport, but this transport only sometimes coincides with remote sources of INP.Equivalent black carbon (eBC) is a quantity derived from aerosol absorption and is the equivalent black carbon mass concentration needed to produce the observed absorption. Other aerosol types such as dust, brown carbon or other organic aerosol might also produce absorption, thus potentially contaminating the small signal we observed. However, absorption at 637 nm by BC is much stronger than for other materials, hence the signal is most likely dominated by BC. BC is produced through a range of combustion processes, including biomass burning, wildfires and fossil fuel combustion, which all occur remote from the central Arctic. Other potential contributors to the absorption signal, such as dust or brown carbon are also remote from the central Arctic. Rigorous procedures were in place to ensure that BC (and other aerosol) from the ship stack did not affect measurements (see methods for details). Therefore, eBC is used here as an indicator of long‐range transport. The literature indicates that BC is a relatively ineffective ice nucleator under mixed‐phase cloud conditions . Similar to the eBC, the surface area and T[INP]= 0.1 6th and the 19th August correlate strongly, but on the 20th August the aerosol surface area decreases by around an order of magnitude without a corresponding decrease in T[INP]= 0.1. This indicates that the variability in INP concentrations at the North Pole is not simply driven by aerosol surface area, rather that some specific component(s) of the aerosol population are ice‐active and these particles are most likely associated with specific sources at latitudes further south than the MIZ.The surface area concentration of the bulk aerosol follows a similar trend to the eBC concentrations, but with a slightly weaker correlation with the 3.4T[INP]= 0.1 > −13.5°C) sampled air masses originating over the Barents and Kara Seas. The next seven highest  filters, in terms of INP activity, sampled air originating from over the Laptev and East Siberian Seas. The next six filters with lower INP activity  sampled air that originated off the eastern coast of Greenland from over both the pack ice and open ocean. The 11 filters with the lowest INP activity  all sampled air which mostly originated from the pack ice adjacent to North America  under high wave conditions .In this section, we assess whether the measured INP concentrations are high enough to initiate a transition from liquid‐dominated clouds to ice‐dominated clouds. Model simulations indicate that on the order of one ice crystal per liter of air is required to remove the bulk of liquid water from an Arctic cloud, whereas lower concentrations still reduce the liquid water path  with the corresponding INP spectra as a rough indicator of primary ice crystal production. This is a crude analysis – a full cloud model would be required to represent ice crystal formation and sedimentation as well as INP recycling and latent heat release, and to predict ice crystal concentration given an INP spectrum – but it does give an indication of what the measured INP spectra might mean for primary ice production in clouds. The highest [INP]ambient will be at the coldest points, that is, the top of the surface mixed layer and the top of the boundary layer (top of the cloud mixed layer), hence this is where we focus this analysis. We assume that ship level measurements are representative of the [INP]ambient at the top of the surface mixed layer and those from SHARK are representative of the top of the boundary layer, the assumption being that these respective layers are individually well‐mixed.The quantity [INP]ambient for the duration of the cruise are shown in Figure ambient in these cases.The temperature minima within the main boundary layer were determined from routine radiosonde profiles with Väisälä RS92 radiosondes, which were made every six hours throughout the entire cruise . The periods of high INP concentration before the 23rd August coincide with periods of higher ambient inversion temperatures, whereas later in the campaign the opposite is the case, which results in a relatively invariant [INP]ambient. Whether this is a coincidence, or if INP concentrations are correlated with ambient temperature, is unclear from this limited data set. However, there may be a physical mechanism behind this apparent correlation. Transport of air to the North Pole from sources further afield will likely result in multiple cycles of cloud formation and dissipation in any one air mass, hence INP active at above the ambient temperature of those clouds will likely activate and be removed via precipitation  should be mixed‐phase, that is, contain a substantial proportion of liquid water with some ice crystals. Observations of the phase of clouds during this campaign are discussed in Vüllers et al. . The mis et al.  are consClouds were multi‐layered around 50% of the time, with many situations identified where seeding of ice from higher, colder clouds into lower clouds might occur. Clouds were regularly observed up to around 8 or 9 km, where temperatures were low enough for homogeneous freezing . We also recommend future studies focus on INP measurements throughout the free troposphere where primary ice production may lead to seeding of ice in lower level clouds.By making measurements of INP spectra both above and within the surface mixed layer of decoupled boundary layers, we found that surface measurements were often not representative of the INPs in the cloud mixed layer. Hence, measurements at altitude, within the cloud mixed layer, are necessary in order to define primary ice production in Arctic mixed‐phase clouds. In addition, our measurements allowed us to estimate the INP concentration active at the temperature of the top of the surface mixed layer and also at the top of the boundary layer. This revealed that, despite massive variability in INP spectra, the INP concentration at ambient temperature was typically less than 0.1 LOverall, it is striking that INP concentrations at the summertime North Pole vary from some of the lowest measured anywhere in the world, to as high as the highest INP concentrations in terrestrial locations rich in biological INPs such as in the UK (O'Sullivan et al., The authors declare no conflicts of interest relevant to this study.Supporting Information S1Click here for additional data file.