These results suggest that the blockage of the host human SUMOylation pathway is very effective for IBV inhibition. We therefore propose that the host SUMOylation pathway is a critical host factor for the IBV virus life cycle. The identification and inhibition of critical host factor(s) provide a novel strategy for future anti-viral therapeutics development, such as IBV and other viruses.The identification and elucidation of host pathways for viral infection are critical for understanding the viral infection processes and novel therapeutics development. Here, for the first time, we discover that the human SUMOylation pathway is essential for the IBV viral life cycle. First, IBV viruses were completely inhibited by a novel SUMOylation specific inhibitor, STE025, discovered from our FRET-based high-throughput screening, and the inhibition was very potent, with IC Influenza A and B viruses  have caused several outbreaks in history, including the recent 2009 H1N1 influenza pandemic, and causes a significant number of deaths every year despite significant advancements in our understanding of the viruses and the availability of vaccines and antiviral agents. In the season of 2017–2018, it is estimated that flu caused approximately 51,000 deaths and 710,000 hospitalizations, making it the most life-threatening infectious disease . Both IBThe efforts to reduce the transmission and pathology of IBV include the introduction of a quadrivalent vaccine that includes both lineages of IBV . These vThere are three FDA-approved neuraminidase inhibitors (NAI) recommended for flu by the CDC: oseltamivir, zanamivir, and peramivir. Most recently, circulating influenza viruses have been susceptible to NAI antiviral medications. However, recent virus isolates from patients show significant drug resistance including IBV ,15,16,17L. monocytogenes, indicating an important role of SUMOylation in promoting these pathogens directly or indirectly [As a pathogen to humans, viruses must engage host factors for their infection and replication, and therapeutics targeting host–virus interactions could present a potentially effective and broad-spectrum treatment modality for viral infections, such as IAV/IBV. Among the interactions of viruses and humans, a post-translation modification (PTM), the SUMOylation, is unique in several aspects. Several genome-wide siRNA or CRISPR screenings have identified that SUMOylation enzyme inhibition or gene knockout decreases the replication of the influenza virus ,23,24. Idirectly . Furtherdirectly ,30,31,32directly   method. Furthermore, we found that the K21R mutation of the M1 protein completely abolishes viral replication, indicating the essential role of SUMOylation for M1 function in the virus cycle. We would like to emphasize that a further understanding of IBV’s dependence on SUMOylation requires further investigation. These efforts suggest that SUMOylation inhibition might be employed for the treatment of IAV and IBV infections.E. coli cells. The plasmids used for the generation of recombinant influenza virus were first described by Fodor et al. [The pET28b (+) constructs for CyPet-SUMO1, UBA2, AOS1, and UBC9 were cloned as outlined in our previous study . The Yper et al. .2 at 37 °C.Human embryonic kidney 293 cells (HEK 293) and Madin–Darby canine kidney cells (MDCK) were cultivated in Dulbecco’s modified Eagles medium  supplemented with 10% fetal bovine serum (GIBCO) and 1% pen strep glutamine 100X (GIBCO) in the incubator with 5% COE. coli cells were transformed with the pET28b (+) constructs encoding CyPet-SUMO1, AOS1, UBA2, UBC9, and YPet-Linker3-M1. The transformed E. coli were plated onto LB plates containing 50 mg/mL kanamycin. A single colony was inoculated into 5 mL liquid LB with 50 mg/mL kanamycin starter culture. Each starter culture was transferred into 1 L 2YT medium with 50 mg/mL kanamycin and grown at 37 °C and 180 RPM for 3 h. The expression of recombinant proteins were induced with 0.35 mM IPTG at 20 °C and 180 RPM overnight. The bacterial cells were harvested the next day at 4 °C and 8000 for 5 min. The bacterial cell pellet was resuspended in 30 mL of a binding buffer . The cell suspension was lysed with an ultrasonic liquid processor . The supernatant was collected after centrifugation at 4 °C and 35,000× g for 30 min. All expression proteins with 6x His-tag were purified by Ni2+-NTA agarose beads . The column was washed sequentially with two column volumes of Wash Buffer 1 (20 mM Tris-HCl pH 7.4 and 300 mM NaCl), two column volumes of Wash Buffer 2 , and one column volume of Wash Buffer 3 , and eluted with an elution buffer . Recombinant proteins were dialyzed overnight at 4 °C in a dialysis buffer . Protein purity was determined by SDS-PAGE after Coomassie G-250 staining , and protein concentrations were determined by a Bradford assay with known amounts of bovine serum albumin  as standards. The concentrations of fluorescent-fusion proteins (CyPet-SUMO1 and Ypet-Linker3-M1) were determined by fluorescence intensities measured on a FlexStationII384 .BL21 DE3 D was determined by setting up a series of reactions with the receptor concentration at 0.20 µM  and a varying concentration of ligand  from 0 to 4 µM. CyPet and YPet binding proteins were mixed and diluted with PBS in a total volume of 60 µL, and this step was repeat three times. The sample was heated up to 37 ℃ and incubated at 15~20 min. Then, all sample mixtures were transferred to a Greiner 384-well plate (Sigma-Aldrich). The fluorescence emissions were measured using FlexstationII384. The emission intensities were measured at three wavelengths: 475 and 530 nm after excitation at 414 nm, and 530 nm after excitation at 475 nm [D was calculated by our previously defined equation, which is shown in Equation (1).The dissociation constant Kt 475 nm . Then th2 in a total volume of 60 mL. The sample mixtures were an added 1 mM ATP were incubated in an Eppendorf tube at 37 °C, and all sample mixtures were transferred to the Greiner 384-well plate (Sigma-Aldrich). The fluorescence emissions were measured using FlexstationII384 . Emission intensities were measured at three wavelengths: 475 and 530 nm after excitation at 414 nm, and 530 nm after excitation at 475 nm [All components of the SUMOylation assay  were combined in the SUMOylation buffer containing 50 mM Tris-HCl pH 7.4, 1 mM DTT, and 4 mM MgClt 475 nm .As described in the DNA constructs section, we fused the CyPet and YPet to the amino termini of the SUMO1 and M1, respectively. The peak wavelengths of excitation and emission were 414 nm/475 nm for CyPet and 475 nm/530 nm for YPet. When the FRET pair (CyPet and YPet) were in close (between 1–10 nm), the excitation of the donor at 414 nm resulted in an energy transfer from the donor to the acceptor, resulting in the quenching of the donor and the excitation of the acceptor. Upon the SUMOylation of YPet-M1 with a CyPet-SUMO1, FRET can occur, resulting in a 530-nm emission given a 414-nm excitation. However, anything that prevents SUMOylation (the absence of ATP or addition of STE025) would result in no increase of the emission at 530 nm.FRET) was used to monitor the formation of the SUMO1-M1 complex. We defined EmFRET as shown in Equation (2) [Total) was differentiated into three fractions: real FRET emission (EmFRET), CyPet direct emission, and YPet direct emission.The real FRET emission (Emtion (2) . To obtaDD) when excited at 414 nm with a ratio coefficient of α = 0.368. The direct emission of YPet at 530 nm was proportional to its emission at 530 nm given a 475 nm excitation (FLAA) with a ratio coefficient of β = 0.029.The direct fluorescence contribution of the CyPet at 530 nm was proportional to its peak emission at 475 nm .g for 5 min, and the MDCK cells were resuspended with 1 mL of DMEM with 10% FBS and 1× Pen-Strep Glutamine (Supplemented DMEM). The 1-mL MDCK suspension was then used to resuspend the HEK293 cells and 500 µL of the cell suspension was distributed among two 10-cm tissue culture-treated plates. A volume of 9.5 mL of supplemented DMEM was added to each plate containing cells, the transfection mixture was added to each plate, and the viruses were generated in double. Twenty-four hours after transfection, the transfection medium was replaced with 1× MEM containing 0.3% BSA, 1% Pen-Strep Glutamine, 0.1% FBS, and 1 mg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin for 72 h at 33°C to produce mature viral particles. The supernatant was then passaged to infect fresh MDCK cells at 90% confluence in a 10-cm plate.The plasmid-based reverse genetic techniques to generate a recombinant influenza virus have been described in the DNA construct section . To gene2 dihydrate, 10.7 mg MgCl2 hexahydrate, 0.42% BSA, and 1× Pen-Strep Glutamine (Gibco Ref: 10378-016). Two hundred milliliters of diluted virus samples was added to each plate and incubated at 33 °C for IBV in a humidified 5% CO2 incubator for one hour with rocking every ten minutes. The supernatant was aspirated and 2 mL of plaquing medium (1× EMEM (Gibco Ref: 11430-030), 0.42% BSA, 1× Pen- Strep Glutamine (Gibco Ref: 10378-016), 10 mM HEPES pH 7.4, 0.1 % sodium bicarbonate, 0.1% dextrose, 1 mg/mL TPCK-trypsin and 0.24% Avicel RC-591 NF) and different concentrations of STE025/PYR-41 were added to each well. The plates were incubated in a humidified 5% CO2 incubator at 33 °C for 72 h. The cells were then fixed with 1 mL of 4% (w/v) paraformaldehyde in PBS for one hour and stained with 1 mL of a solution containing 1% crystal violet (w/v) in 10% methanol (v/v).Confluent MDCK cells were seeded into each well of 6-well tissue culture-treated plates. After a 24-h culture, the cells were pretreated with different concentrations of STE025/PYR-41 for 24 h. Viruses were serially diluted in a total volume of 300 mL in a medium consisting of 1× PBS, 6.11 mg CaCl4 MDCK cells per well were seeded in a 96-well plate one day before performing the Natural red assay. The experiment was divided into four conditions . After a 24-h culture, half of the plate was pretreated with different concentrations of specific drug overnight. The culture media was removed and washed with 1X PBS. Virus samples were diluted in 1X PBS with Pen strep glutamine, CaCl2-2H2O/MgCl2-6H2O 100X, and 35% BSA. The appropriate dilution, which made the MOI reach 0.01, was added to the corresponding well and incubated for 72 h at 33 °C and 5% CO2. The culture media were removed and washed with a 1X washing buffer. The plates were then stained with a 100X neutral red staining solution for approximately 2h at 37 ℃ in a 5% CO2 incubator. The neutral red staining solution was removed by complete aspiration, and the cells were rinsed with a 1X washing buffer to remove the residual dye. The washing buffer was completely removed, and the incorporated neutral red was eluted with a 1X solubilization solution for 30 min. The dye content in each well was quantified using FlexstationII384  at a wavelength of 540 nm. The dye content in each set of wells was converted to a percentage of the dye range present between the virus control and the untreated control wells using a Microsoft Excel computer-based spreadsheet.The Neutral Red Cell Cytotoxicity Assay Kit  was used in this experiment. A total of 1 × 10Many host-viral interactions have been reported before, including those in influenza viruses and coronaviruses ,44,45. IWe first determined whether the IBV was sensitive to the SUMOylation-specific inhibitor, STE025. Due to the lethality of SUMO E1, E2, and multiple E3 gene-knockout mice, it is not feasible to determine the role(s) of SUMOylation in many physiological or pathological processes. We, therefore, developed a FRET-based high-throughput screening to discover small molecule inhibitor(s) for SUMOylation to dissect the roles of SUMOylation. After screening for more than 220,000 compounds and validating hits, we identified a specific SUMOylation inhibitor, STE025, but not for ubiquitination or NEDDylation . We first tested the potential antiviral activity of STE025 for IBV in a plaque assay. Significantly, the STE025 could almost completely inhibit IBV growth at the concentration of 3.5µM, indicating an excellent inhibitory activity of the STE025 for IBV replication A. This rThe SUMOylation is a Ubiquitin-like cascade that adds SUMO peptide to the target proteins to regulate their activities and sometimes to compete with the Ubiquitin-mediated protein degradation. To determine whether IBV replication also requires ubiquitination, we used a ubiquitination small molecule inhibitor, PTR-41, which explicitly inhibits the Ubiquitin-Activating Enzyme (E1) . Our res50 of the STE025 inhibition for the SUMOylation is approximately 0.8 µM in a biochemical SUMOylation reaction. A cell-based assay of cell death induced by IBV was set up to determine the potency of the STE025 for IBV inhibition using the neutral red cell cytotoxicity assay. MCDK cells were plated in a 96-well plate the day before a pre-treatment of the STE025 or as a control, PTY-41. The cells were infected with IBV viruses for three days before the cell toxicities were determined. Compared with the cells infected by IBV (red line), the STE025 rescued IBV-infected cells from death in a dose-dependent mode  and purified through the Ni-His affinity column. Then, in a fixed concentration of the FRET donor, the CyPet-Ubc9 or the CyPet-PIAS1, different concentrations of the FRET acceptor, the YPet-M1, were titrated. We have developed an algorithm to extract the absolute FRET signal, which corresponded to the interactions, from the total fluorescence signal [FRET) for Ubc9 and PIAS1 with M1 showed good sigmoidal curves  of the IAV M1 protein through a combination approach of FRET-SUMOylation-MS, we found that several sites of IAV M1 were SUMOylated, including Lys 21, Ly35, Lys 187, Lys 230, Lys 242, and Lys 252 . We mutated each of the above Lys in the IAV M1 gene, and we found that the Lys21Arg mutation completely abolished viral replication. IBV is similar to IAV in many aspects, from the genome to transmission and pathology. We speculated that viral interactions with the host might be similar to some degree. We first aligned the IBV M1 and IAV M1 proteins and found a very low similarity A. Howeve50 in in vitro SUMOylation assay, suggesting a good potential in in vivo applications. The mechanism underlining the viral inhibition by the SUMOylation inhibitor STE025 is at least partly due to the requirement of SUMOylation of the IBV M1 protein. The IBV M1 protein is recognized by the SUMOylation E2 conjugation enzyme and E3 ligase at very high affinities, both around 0.2 µM in our qFRET assay, suggesting the SUMOylation of M1 under in vivo physiological conditions. The detailed functional requirement of SUMOylation for the M1 protein needs to be further explored, but this protein is known to play essential roles in viral RNA trafficking and in viral assembly. In addition, the clinical data showed that children were more susceptible and had a higher rate of hospitalization when infected with IBV than IAV, reflecting different pathological processes and immune responses caused by IBV infection [Viruses infect hosts, such as humans, by taking advantage of host factors for their life cycles. The roles of these factors are different depending on whether they can be replaced by other host factors or not. Here, for the first time, we determine that the human SUMOylation pathway is essential for the IBV life cycle. IBV virus replication can be inhibited by the novel SUMOylation inhibitor, STE025, in a plaque assay. The STE025 has a very high inhibition potency, at about 0.1 µM in the rescuing IBV-induced cell death assay. This potency is higher than its ICnfection . In a cenfection . AlthougThe SUMO E3 ligase family of PIAS was first discovered through a yeast two-hybrid screening for its interactions with STAT proteins. The overexpression of PIAS genes inhibits cytokine signaling, which is critical in innate immune responses against viral infections ,57,58. T−4–10−6 substitutions/bp/cell infection) mainly due to the lack of proofreading activities of their RNA-dependent RNA Polymerases (RdRP) and RNA-dependent DNA Polymerases (RdDP) or Reverse Transcriptases (RTase), [−9–10−10 substitutions/bp/cell division), mostly due to both the proofreading activity of DNA polymerase and the mismatch repair pathways of host cells [Targeting host factors for the treatment of viral infections can bring potential additional important benefits of reduced drug resistance. There may be several benefits to targeting host factors. First, the viral genomes, particularly ssRNA viruses, such as influenza viruses, have a very high mutation rate (10(RTase), ,72,73,74st cells ,76. Therst cells .A new approach to targeting host factors for anti-pathogens has emerged as a new strategy for anti-viral therapeutics ,44,74,78The SUMOylation inhibitor and its application in anti-viruses and anti-cancers was patented by the University of California.


OHS motifs representing an immunodominant IgE epitope. So far, recombinant Ara h 2 has been produced in Escherichia coli, Lactococcus lactis, Trichoplusia ni insect cell, and Chlamydomonas reinhardtii chloroplast expression systems, which were all incapable of proline hydroxylation. However, molecular diagnosis of peanut allergy is performed using either natural or E. coli-produced major peanut allergens. As IgE from the majority of patients is directed to Ara h 2, it is of great importance that the recombinant Ara h 2 harbors all of its eukaryotic posttranslational modifications. We produced hydroxyproline-containing and correctly folded Ara h 2 in the endoplasmic reticulum of leaf cells of Nicotiana benthamiana plants, using the plant virus-based magnICON® transient expression system with a yield of 200 mg/kg fresh biomass. To compare prokaryotic with eukaryotic expression methods, Ara h 2 was expressed in E. coli together with the disulfide-bond isomerase DsbC and thus harbored disulfide bridges but no hydroxyprolines. The recombinant allergens from N. benthamiana and E. coli were characterized and compared to the natural Ara h 2 isolated from roasted peanuts. Natural Ara h 2 outperformed both recombinant proteins in IgE-binding and activation of basophils via IgE cross-linking, the latter indicating the potency of the allergen. Interestingly, significantly more efficient IgE cross-linking by the N. benthamiana-produced allergen was observed in comparison to the one induced by the E. coli product. Ara h 2 from N. benthamiana plants displayed a higher similarity to the natural allergen in terms of basophil activation due to the presence of hydroxyproline residues, supporting so far published data on their contribution to the immunodominant IgE epitope. Our study advocates the use of N. benthamiana plants instead of prokaryotic expression hosts for the production of the major peanut allergen Ara h 2.Peanut allergy is a potentially life-threatening disease that is mediated by allergen-specific immunoglobulin E (IgE) antibodies. The major peanut allergen Ara h 2, a 2S albumin seed storage protein, is one of the most dangerous and potent plant allergens. Ara h 2 is posttranslationally modified to harbor four disulfide bridges and three hydroxyprolines. These hydroxyproline residues are required for optimal IgE-binding to the DPYSP Arachis hypogaea) harbor 18 different allergenic proteins , including the highly potent 2S albumin Ara h 2, which makes up 6–9% (1.6–2.3 g/kg) of the total seed protein . Ara h 7 has been recently described to have partial cross-reactivity with Ara h 2  antibodies, affects up to 30% of the world population  or recombinant (r) allergens are preferable for at-risk patients. Isolated natural allergens have been largely replaced by recombinant allergens produced in prokaryotic hosts, mainly in Escherichia coli  assay  and latex  by inoculating plant leaves with in vitro transcribed viral RNA, which were all shown to bind IgE from allergic patients -based magnICON® expression vectors  a 5′-provector (pICH20155) encoding viral genes necessary for plant transformation, (ii) a 3′-provector encoding the gene-of-interest (pICH31070), (iii) a separate plasmid encoding the PhiC31 integrase (pICH14011) that enables in planta recombination of both modules . A combination of plasmids encoding transgenes framed by left and right T-DNA borders  was used for N. benthamiana and synthesized and ligated into the pEX-A128 cloning vector . The subcloning was performed via BsaI restriction sites, following the Golden Gate cloning protocol  cells (recently renamed to Rhizobium radiobacter) were transformed with either pICH14011, pICH20155, or pICH31070. The transformed bacteria were grown in YEB medium  with the antibiotics kanamycin (50 mg/L) and rifampicin (25 mg/L) to an OD600 of 1.5–2 at 28°C. The cultures were then centrifuged at 8,500 × g for 1 h at 4°C. The pellets were resuspended in infiltration buffer , and the OD600 was adjusted to 1.0 by mixing three transformants in a 1:1:1 ratio. The leaves of 7-week-old N. benthamiana plants  were infiltrated using a syringe without a needle with the mixture of agrobacteria suspensions, carrying either of the three plasmids.Electrocompetent g for 30 min at 4°C. Total soluble and insoluble fractions containing rAra h 2 were prepared as follows. An aliquot from the homogenized leaf suspension was taken for mixing with the reducing sample buffer , heated at 95°C and loaded as the total fraction. The rest of the homogenized leaf suspension was centrifuged at 14,000 × g for 15 min. An aliquot from the supernatant was mixed with the reducing sample buffer, heated at 95°C and loaded as the soluble fraction. The pellet was mixed with the reducing sample buffer, and an aliquot was taken for heating at 95°C prior to loading.Infected leaves were collected 10 days post-infiltration, snap frozen in liquid nitrogen and ground using a mortar and a pestle. One gram of ground-up leaves was resuspended in 3 mL of the extraction buffer , containing a cocktail of protease inhibitors . Proteins were extracted by vigorously vortexing the ground-leaf suspensions at 4°C. Additional incubation of the total leaf suspensions overnight at −20°C helped with cell lysis. Thawed suspensions were cleared by centrifugation at 14,000 × 2HPO4, 136 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.4) before measuring the concentration using the bicinchoninic acid assay (BCA) .All chromatographic steps were performed using an FPLC system. The supernatant of the crude extract was dialyzed against the loading buffer . Filtered samples were applied to a Ni-NTA-agarose column , and rAra h 2 was eluted by adding 25 mM to 100 mM imidazole. As a second purification step, anion exchange chromatography was performed using a MonoQ column . rAra h 2 was loaded onto the column in buffer A  and eluted with increasing NaCl concentrations, recovering the protein at around 220 mM. Pure proteins were concentrated and dialyzed against phosphate-buffered saline (PBS) . For Western blot analysis, unstained gels were blotted onto nitrocellulose membranes  at 400 mA constant for 1 h at room temperature. Membranes were incubated with the anti-Ara h 2 monoclonal antibody 1C4   or with sera from patients (1:10). An anti-mouse IgG + IgM conjugated to alkaline phosphatase (AP)   or anti-human IgE conjugated to AP  were used as secondary antibodies, respectively. 5-Bromo-4-chloro-3-indoyl phosphate (BCIP) and nitro blue tetrazolium chloride (NBT) were used for developing the signals. For percentage of soluble protein analysis, the immunoblot was analyzed using ImageJ 1.8.0  with 5 mM imidazole and an EDTA-free protease inhibitor cocktail  and lysed three times by high-pressure cell disruption. Lysed pellets were centrifuged at 18,000 × g for 1 h at 4°C. To remove the bacterial DNA, the supernatant was treated with 1:1,000 Biocryl BPA-1,000 . Histidine-affinity chromatography purification was performed as explained before, and rAra h 2 was recovered at around 50 mM imidazole. Anion-exchange chromatography was performed as explained before, recovering the protein at around 170 mM NaCl. Pure proteins were concentrated and dialyzed against PBS before assessing the concentration using the BCA .The wild-type sequence of Ara h 2.0201 was subcloned into the pMWHisN expression vector, an in-house designed T7 expression vector based on pMW172  were measured by ImmunoCAP . A value higher than 0.35 kUA/L indicated a positive result. Skin-prick tests were performed with commercial peanut extracts, and results were considered positive when the wheal diameter was ≥3 mm compared with the saline control. Three non-atopic controls  without a history of type 1 allergy, and three atopic patients  with allergies other than peanut were recruited as controls (*Power 3.1.9.4 software. The sample size was found sufficient to detect differences between groups of about 0.6 standard deviation with a power of 90%.Twenty adults (aged 18–54 years) with a medical history of peanut allergy were recruited at the Department of Dermatology, University Hospital St. Pölten . Sera frcontrols . Power c3, pH 9.5). After blocking with 3% skimmed milk powder in Tris-buffered saline (TBS)  with 0.5% Tween-20, wells were incubated overnight at 4°C with sera from 20 patients with peanut allergy, three non-atopic patients, or two atopic patients without peanut allergy (diluted 1:10). An anti-human IgE conjugated to AP  was used for detection, and the reaction was developed using p-nitrophenyl phosphate tablets . The plates were measured after 20 min at OD405 , using the SparkControl Magellan 2.2 software. The plotted values were calculated by subtracting the mean value obtained from all negative controls plus three times the standard deviation from the raw data.For ELISA, pure proteins were coated onto microtiter plates overnight at 4°C at 2 μg/mL protein in sodium carbonate buffer (50 mM NaHCOBasophil activation test (BAT) using human IgE-receptor expressing rat basophil leukemia cells (RBL) were performed following a published protocol  and two-tailored P-values. All analyses were done using GraphPad Prism 9.0.0 .The Friedman test with the Dunn posttest was performed to compare IgE-binding and basophil activating performance of different proteins. The results from the ELISA and RBL assays were correlated with the Ara h 2-ImmunoCAP values of the patients by computing Spearman's rank correlation coefficients  to alter the canonical N-glycosylation site  was performed to identify contaminants and to detect PTMs. rAra h 2 was detected with 89.6% sequence coverage and with the highest score among all detected proteins, indicating high confidence of the match  than the nAra h 2 . Six out of seven hydroxyproline residues passed the manual confirmation (N. benthamiana (PRIDE repository dataset identifier: PXD027015).Mass spectrometry analysis of rAra h 2 , which was significantly higher than both N. benthamiana-produced  and E. coli-produced allergens .The IgE-binding capacities of rAra h 2 from gE ELISA . In a cogE ELISA , IgE of 405 values for the recombinant Ara h 2 proteins. Notably, patients P7, P9, P17, P18, and P20 had mean OD405 values for IgE-binding to plant-made rAra h 2 that were higher than the values obtained for the E. coli recombinant protein . Additionally, we investigated the correlation between ELISA results for all three proteins and sIgE values of patients measured to Ara h 2 by ImmunoCAP .Patient-specific IgE recognition of all Ara h 2 proteins is shown in  protein . In contmmunoCAP . SignifiN. benthamiana) and to nAra h 2 correlated significantly with the ImmunoCAP values . On the other hand, there was no significant correlation between sIgE concentrations of patients and the ratio of IgE-binding to rAra h 2 (E. coli) and to nAra h 2 . IgE from patients with lower sIgE concentrations (ImmunoCAP <50 kUA/L) bound to nAra h 2 better than to either of the recombinants , the ratio of IgE-binding to rAra h 2 (N. benthamiana) and to rAra h 2 (E. coli) was >1 for three patients, who had lower sIgE levels  or the rAra h 2 from E. coli  almost twice as much as the one from E. coli  (median SI = 4.0) nor rAra h 2 (E. coli) (median SI = 3.5) were significantly different from the nAra h 2 (median SI = 4.6)  of 20 failed to efficiently sensitize the cells (shown by an SI <2 upon stimulation with anti-IgE as positive control). Data obtained from these patients are not included in I = 4.6) . Interesntration .N. benthamiana) and nAra h 2 and the sIgE values of the patients . However, the ratio of the SIs of rAra h 2 (E. coli) and nAra h 2 decreased significantly when the sIgE levels of patients increased . In contrast, a strong positive correlation was observed when the ratio of SI for rAra h 2 (N. benthamiana) and for rAra h 2 (E. coli) was plotted against the sIgE levels of patients .To investigate these observations together with sIgE values of patients, we plotted ratios of SI for each allergen (at 0.1 ng/mL) against the ImmunoCAP values . There wN. benthamiana leaf cells by employing a plant virus-based transient expression system and studied its biochemical and immunological characteristics. Agrobacterium tumefaciens-mediated expression in N. benthamiana plants yielded 200 mg rAra h 2 per kg biomass within 10 days. Site-specific proline hydroxylation was observed within the DPYSPS repeats of rAra h 2 expressed in N. benthamiana. Unlike the natural protein, which has three hydroxyprolines at the SPS sequences, N. benthamiana-made rAra h 2 was modified at six proline residues within the SPS, DPY, and SPY sequences. The involvement of these PTMs in IgE-binding and cross-linking was investigated using immunological assays. For this purpose, N. benthamiana-produced Ara h 2 was compared with the hydroxyproline-free recombinant expressed in E. coli and with the natural allergen isolated from peanuts. Natural Ara h 2 outcompeted both of the recombinant proteins in all immunological assays used. Although neither of the recombinant proteins outperformed the other in ELISA, IgE-cross-linking results obtained from BAT revealed a remarkable superiority of the N. benthamiana-produced allergen over the E. coli product. Thus, a higher similarity of the plant-made rAra h 2 to the natural protein was shown. The E. coli-produced protein was co-expressed with the disulfide-bond isomerase DsbC, therefore, harbored disulfide bridges , followed by expression in the cytosol (6.5 mg/kg) and the ER (0.65 mg/kg)  out of 19 patients (21%) reacted to the recombinantly produced Ara h 2 proteins almost as well as the natural protein . The IgE product . Lower-s product , are mor product . InteresN. benthamiana activated basophils more efficiently than the E. coli recombinant already at the lowest concentration .The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via PRIDE partner repository  and conducted in accordance with the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.ÖÜ and TK designed and performed experiments. ÖÜ, TK, CR, and HB analyzed the data. VM and NL assisted in the experiments. AT produced reagents and performed experiments. CH and WH provided sera from patients. HB provided input and supervised experiments. ÖÜ and HB interpreted the data and wrote the manuscript. All the authors read the manuscript.This study was supported by the Austrian Science Fund (FWF) grants MCCA W1248-B30 and P 30936-B30 and the Danube Allergy Research Cluster project P06 funded by the Country of Lower Austria.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.


Foil or latex glove interpositions did not prevent screw stripping. Thus, in the current experimental study, T-handle screwdriver or hex key wrench usage decreased the incidence rate of screw stripping during removal compared to straight-handle screwdriver use.There have been several reports about the difficulties in removing 3.5-mm titanium locking screws from plates due to the stripping or rounding of the hexagonal screw socket. We investigated whether stripping the locking screw sockets can be prevented by using different screwdrivers or interposing materials into the socket during removal. We overtightened 120 3.5-mm titanium locking screws  equally into locking plates on sawbone tibia models, applying a uniform torque of 4.5 Nm, exceeding the recommended torque of 1.5 Nm. Twenty screws each were removed using a straight-handle 2.5-mm screwdriver, T-handle screwdriver, hex key wrench, and straight-handle screwdriver with a non-dominant hand. In addition, 20 screws were removed using foil from a suture packet inserted into the screw socket or using parts of a latex glove inserted into the screw socket. The incidence rates of screw stripping using the straight-handle screwdriver, T-handle screwdriver, hex key wrench, non-dominant hand, foil interposition, and latex glove interposition were 75%, 40%, 35%, 90%, 60%, and 70%, respectively. When a T-handle screwdriver or hex key wrench was used, the probability of screw stripping was 4.50 times (odds ratio = 4.50, 95% confidence interval = 1.17 to 17.37,  However, titanium is prone to cross-threading or cold welding under strong metal-to-metal contact9. There have been several reports about the difficulties in removing 3.5-mm titanium locking screws from the locking plates after healing of the fractured bone16. Removal is challenging when the hexagonal socket of the screw is stripped, rendering the drivers useless. Various techniques have been suggested to remove stripped locking screws18. However, once the screw is stripped, the surgery duration is prolonged, despite the broken screw or stripped screw removal techniques, which increases the possibility of complications such as more bleeding, infection, neurovascular injury, and even iatrogenic fractures11. Therefore, the prevention of screw stripping is essential.Locking plates can provide high fixation stability for reliable healing in various fractures. These plates are usually manufactured from titanium alloys with higher biocompatibility, higher fatigue strength, and lower risk of infection and are isoelastic with the bone compared to stainless steel19. Some authors suggest the insertion of aluminum foil or part of a sterile glove into the screw socket to increase the friction between the driver and the socket; nevertheless, these tips are anecdotal, and none have been proven effective22. Currently, star-shaped  sockets, which are less prone to stripping, are now widely used for locking screws23. However, conventional locking screws with hexagonal sockets, widely used in the past and still used, may require removal someday.Appropriate use of aiming sleeves and torque-limiting screwdrivers is important to prevent cross-threading or overtightening of the screws during insertion. However, there are few suggestions that can be followed during screw removal to prevent screw strippingWe performed an experimental study on sawbone tibia models to determine whether the incidence of stripping of the hexagonal socket of 3.5-mm titanium locking screws  can be minimized by using a T-handle driver with a relatively larger dimension of the driver tip or a hex key wrench. We also tested whether there was a difference in the incidence of screw stripping by using the dominant hand and the non-dominant hand. In addition, we tested whether the interposition of the aluminum foil or parts of the latex glove into the screw socket effectively reduces the incidence of screw stripping.We prepared 6 sawbone tibia models and 12 3.5-locking compression plates  with 10 holes each and 120 3.5-mm titanium locking screws (Depuy Synthes). Two plates were attached to each sawbone tibia model. To overtighten and jam the locking screws into the locking plates with the same amount of torque, a torque driver with the torque limit set at 4.5 Nm was used. A total of 120 screws were overtightened by applying a torque of 4.5 Nm, which exceeded the recommended torque of 1.5 Nm for the 3.5-mm locking screw insertion , a T-handle screwdriver , and a hex key wrench  were used to remove 20 screws each  and the use of different screwdrivers or the interposition of materials. A chi-square test was performed on the two-way contingency table with the row variable set as the use of different screwdrivers or the interposition of materials  and the column variable set as the binary outcomes (stripped and irremovable screws/removed screws). The use of straight-handle screwdrivers was used as the reference. We calculated the odds ratio and its 95% confidence interval (95% CI) on this two-way contingency table to measure the association between the use of different screwdrivers or interposition of materials and prevention of screw stripping. We used Fisher's exact test to analyze whether the non-dominant hand was associated with a high incidence of screw stripping. Statistical significance was set at p < 0.05. Statistical analysis was performed using SPSS version 22.0 .We performed a pilot study on 20 3.5-mm titanium locking screws jammed into the locking plate to determine the appropriate sample size for the study. As a result, 8 out of 10 (80%) and 3 out of 10 (30%) screws were stripped using the straight-handle driver and the T-handle screwdriver, respectively. Assuming 95% power and a 0.05 significance level, 20 samples in each group were calculated to detect a decrease in the incidence of screw stripping from 80% in the control group to 30% in the experimental groupThis study was approved by the IRB of Hallym University Kangnam Sacred Heart Hospital (IRB-2020-12-015). The study did not involve humans or animals.The results are presented in Table p = 0.03), and 5.57 times  when the T-handle screwdriver and the hex key wrench were used, respectively, compared to that using the straight-handle screwdriver  (Tables p = 0.002), and 16.71 times  when the dominant hand was used with the T-handle screwdriver and the hex key wrench, respectively, compared to that using the non-dominant hand with the straight-handle screwdriver  Tables ,3. The p25. However, there are reports of difficulties in removing 3.5-mm titanium locking screws due to stripping of hexagonal sockets16. This may be because titanium is more malleable than stainless steel, and the difference in the mechanical resistance between the stainless-steel screwdriver and the titanium screw may lead to stripping of the titanium screw. Titanium has a high degree of biocompatibility, which leads to increased osteointegration and thus a higher risk of screws becoming encased26. Furthermore, LCP is used for the minimally invasive plate osteosynthesis technique. Hence, when the locking screw hole of the plate is not well exposed, the locking screw may be inserted at the wrong angle and jam into the plate22. During screw removal, the screwdriver may sometimes engage with the screw at an incorrect angle, or the driver tip may not be fully engaged in the screw socket before the screwdriver is turned. The surgeon can feel the first slippage of the screwdriver inside the socket. A biomechanical study demonstrated up to a 50% decrease in the maximal torque values after a single event of screwdriver slippage13. Although the straight handle is easy to rotate when there is no considerable resistance, as in screw insertion, when a high torque for the counterclockwise rotation force is required to remove a jammed locking screw, the straight-handle screwdriver is relatively thin for a full grip compared to the T-handle. The axis of the straight-handle driver may not be in alignment with the axis of the screw when considerable wrist movement is required in addition to finger movement to rotate the screwdriver. This may cause the tip of the driver to deviate inside the screw socket  has been introduced as a new standard of AO plating osteosynthesis. It is widely used for various fractures, such as comminuted tibia fractures, or even in joint sparing salvage surgery after articular oncological resections13.However, for convenience during screw insertion, most manufacturers provide a straight-handle driver; moreover, the 2.5-mm straight-handle screwdriver (Depuy Synthes) is commonly provided by the scrub nurse to remove 3.5-mm titanium locking screws (Depuy Synthes). The diameter, length, and design of the 2.5-mm hexagonal driver tip can differ between the products by different manufacturers. Many manufacturers provide a screwdriver shaft that can be connected to the power tool for power insertion of the screws. This screwdriver shaft can be connected to the T-handle. We recommend that surgeons source the largest available 2.5-mm driver tip with sharp edges by testing the engagement of the driver tip in the screw socket, checking how firmly it can hold the screw, and reserving it for screw removal. In some situations, surgeons may have to change their position to use their dominant hand for screw removal. But some may prefer to use their non-dominant hand rather than change their position to use the dominant hand. Because one inadvertent missed turn of the screwdriver with the non-dominant hand can ream the socket, leading to screw stripping, we recommend that surgeons use their dominant hand for screw removal on all occasions17. Strong cortical bone ingrowth at the terminal flutes of the screw can cause screw stripping; however, this situation could not be simulated with sawbone models27. Second, the amount of torque that leads to screw stripping can be different in different cases. However, for the experimental study, we overtightened all locking screws by applying a 4.5-Nm torque. The results may be different, with different torques applied to overtighten the screw. Third, although the interposition of the foil or parts of the latex glove did not significantly decrease the incidence of screw stripping in the current study, it may potentially be effective with different torques applied on the screws. However, we believe that the interposition of the material can impede the engagement of the driver tip into the screw socket to its full depth, which can lead to a decrease in the contact area  to remove3.5-mm titanium locking screws (Depuy Synthes). There are several screw removal devices that are designed to remove stripped screwsThe use of a T-handle screwdriver reduced the incidence of screw stripping by 4.50 times compared to the use of a straight-handle screwdriver.The use of a hex key wrench decreased the incidence of screw stripping by 5.57 times compared to the use of a straight handle screwdriver;The interposition of foil or parts of the latex glove into the screw socket did not decrease the incidence of screw stripping.There have been several reports about the difficulties in removing 3.5-mm titanium locking screws from the locking plate due to the stripping of the hexagonal screw socket. However, there is a lack of research on methods to prevent screw stripping during screw removal. We hypothesized that screw stripping can be prevented by using different screwdrivers or interposing materials into the socket during removal. We performed an experimental study on swabone tibia models, and tested the incidence rate of screw stripping using a straight-handle screwdriver, T-handle screwdriver, hex key wrench, non-dominant hand, foil interposition, and latex glove interposition. In the current experimental study;A future study on the surface morphology of stripped screws using emission scanning electron microscopy may enable a more accurate analysis of the effects of different screwdrivers on screw stripping. Furthermore, clinical application results are required to confirm the above findings.


The type of inhibition can also be determined from the curve of the degree of inhibition by considering the shift of the inhibition curves. Amperometric experiments were performed with a biosensor polarized at the potential −0.15 V vs. Ag/AgCl and using 0.1 M phosphate buffer (pH 6.8) as an electrolyte. Under optimized conditions, the proposed biosensor showed a linear amperometric response toward catechol detection from 0.5 µM to 38 µM with a detection limit of 0.35 µM (S/N = 3), and its sensitivity was 66.5 mA M−1 cm−2. Moreover, the biosensor exhibited a good storage stability. Conversely, a novel graphical plot for the determination of reversible competitive inhibition was represented for free tyrosinase. The graph consisted of plotting the half-time reaction (t1/2) as a function of the inhibitor concentration at various substrate concentrations. This innovative method relevance was demonstrated in the case of kojic acid using a colorimetric bioassay relying on tyrosinase inhibition. The results showed that the t1/2 provides an extended linear range of tyrosinase inhibitors.An amperometric biosensor based on tyrosinase, immobilized onto a carbon black paste electrode using glutaraldehyde and BSA was constructed to detect competitive inhibitors. Three inhibitors were used in this study: benzoic acid, sodium azide, and kojic acid, and the obtained values for fifty percent of inhibition (IC This means that at a high concentration of inhibitor , the degree of inhibition tends to deviate from linearity.In this regard, terature ,45. We pterature  that the50 values for all tested inhibitors against tyrosinase were summarized in 50 value increases with increasing substrate concentration. Given this, the use of a high concentration of substrate will not produce significant inhibition responses.Further experiments of tyrosinase inhibition were carried out for benzoic acid and sodium azide. The ICThe inset of I%) was obtained by varying the concentration of inhibitors at a fixed substrate concentration of 20 µM  was calculated from the degree of inhibition curves by plotting the percent inhibition versus inhibitor concentration. The IC50 values were determined to be 119 µM, 1480 µM, and 30 µM for benzoic acid, sodium azide, and kojic acid, respectively.The concentration that inhibited 50% of the catechol signal /t versus 1/t 2.3 log(S0/S) is used to determine the kinetic parameters Km (slope) and Vmax (intercept).The curve of  represents the Michaelis–Menten equation:Vi presents the velocity in the inhibitor’s presence, I is the concentration of the inhibitor, and Ki presents the dissociation constant of the enzyme-inhibitor (EI). The integrated Michaelis–Menten equation for competitive inhibition is: t’) is not linearly related to the inhibitor concentration (I), since the log conversion rate (S0/S) varies with time. However, the measurement at the time required to achieve 50% of the substrate conversion in the presence of an inhibitor (t’½) leads to a linear relation with the inhibitor concentration.As mentioned in this integrated rate Equation (6), time  will affect the value of the intercept (A), but the value of the slope  and 10), any change of the substrate concentration (, any chaHerein, we describe for the first time, an easy-to-use graphical plot for the determination of a reversible competitive inhibition type. Thus, the use of a progress curve for inhibitor determination allows for a plot of the time required to achieve 50% of a substrate conversion versus inhibitor concentrations. It is important to note that using the integrated progression curve equation allows for an extended linear range, which is highly required for inhibitor determination in undiluted samples. The proposed graph was successfully applied for tyrosinase using L-tyrosine as a substrate and kojic acid as an inhibitor. Furthermore, the diagnosis of competitive inhibition can be performed by plotting the degree of inhibition versus inhibitor concentration. Amperometric tyrosinase biosensor was successfully employed using the degree of inhibition to determine benzoic acid, sodium azide, and kojic acid at the micromolar level.


The left ventricular noncompaction cardiomyopathy (LVNC) is a rare subtype of cardiomyopathy associated with a high risk of heart failure (HF), thromboembolism, arrhythmia, and sudden cardiac death.The proband with overlap phenotypes of LVNC and hypertrophic cardiomyopathy (HCM) complicates atrial fibrillation (AF), ventricular tachycardia (VT), and HF due to the diffuse myocardial lesion, which were diagnosed by electrocardiogram, echocardiogram and cardiac magnetic resonance imaging. Peripheral blood was collected from the proband and his relatives. DNA was extracted from the peripheral blood of proband for high-throughput target capture sequencing. The Sanger sequence verified the variants. The protein was extracted from the skin of the proband and healthy volunteer. The expression difference of desmocollin2 was detected by Western blot.DSC2 gene encoding an important component of desmosomes was detected by targeted capture sequencing. The western blots showed that the expressing level of functional desmocollin2 protein (~ 94kd) was lower in the proband than that in the healthy volunteer, indicating that DSC2 p.K47Rfs*2 obviously reduced the functional desmocollin2 protein expression in the proband.The novel heterozygous truncated mutation (p.K47Rfs*2) of the DSC2 p.K47Rfs*2 remarkably and abnormally reduced the functional desmocollin2 expression, which may potentially induce the overlap phenotypes of LVNC and HCM, complicating AF, VT, and HF.The heterozygous  MYH7), protein-binding protein C myosin (MYBPC3), tropomyosin alfa (TPM1), myocardial actin (ACTC1), troponin T (TNNT2), and cardiac troponin I. The less common mutations of genes inducing LVNC include Z-band protein mutations, such as Cypher/ZASP cytoskeleton protein, alpha-distrobrevin (DTNA), calcium transport proteins, calsequestrin, phospholamban, membrane proteins (A/C lamina), para-zinc-finger transcription factor [The left ventricular noncompaction cardiomyopathy (LVNC) is a rare subtype of cardiomyopathy (CM) and is characterized by predominant left ventricular trabeculations with deep intertrabecular recesses and thinning of the compact epicardium . LVNC can factor , and mitAll participants signed informed consent. All procedures performed in the study involving human participants were in accordance with the Declaration of Helsinki and ethical standards and were approved by the Medicine Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (No. 2020K216-1).In accordance with previous studies , 9, LVNCImaging was acquired using the 1.5 T MR imaging system  by using a 6-channel phased-array coil. After scout and reference scans, functional and geometric assessments were achieved using ECG-gated, cine steady-state-free precession images in standard long-  and short-axis orientations with full ventricle coverage from basis to apex. Left and right ventricular outflow tract cine sequences were also obtained. Cine images with a temporal resolution of approximately 40 ms were obtained. Additional pulse sequences, including T1WI and T2WI of blood-suppressed double inversion recovery fast spin-echo, were acquired in a standard location before contrast administration. Then, the dynamic enhancement was performed during the administration of 0.2 mmol/kg contrast agent  with 50 frames. Late gadolinium-enhanced imaging was performed with 20 min delay and phase-sensitive inversion recovery sequence to detect myocardial scarring or fibrosis. CMRI analyses were performed using SigoVia (Siemens). The functional and geometric left ventricular indices, including left and right ventricular end-diastolic volumes, LVEF and right ventricular ejection fraction, indexed left ventricular compacted mass with papillary muscles, indexed left ventricular compacted and noncompacted mass, indexed left ventricular noncompacted mass, and their ratio was determined. In addition, the end-diastolic extent of compacted and noncompacted myocardium and their ratio were measured in one long axis geometry.http://sift.jcvi.org/www/), PolyPhen-2 , and MetaSVM [Skin biopsies were collected from the upper left arm of the proband and healthy volunteer. Protein was extracted from tissues. Peripheral blood was collected from the proband and his relatives. DNA was extracted  from the peripheral blood of proband for high-throughput target capture sequencing using Gene fragment capture chip  and MGIseq-2000 sequencer . The panel of common risk genes associated with CMs and arrhythmias Table  was dete MetaSVM . Variant MetaSVM  followin MetaSVM . Variant MetaSVM .Table 1SAccording to ACMG standards and guidelines, all variants have been reclassified for interpreting sequence variants as P, LP, VUS, LB, or B . The PM2DSC2 p.I520T was screened again using Sanger sequencing  in the other members of the family. The mutation of DSC2 p.K47Rfs*2 was screened again using the TA cloning technique in the other familial members. The purified PCR product was directly linked with pClone007 Versatile Simple Vector  by TA cloning technique and transformed into Escherichia coli (DH5α). The extracted plasmids were sequenced with ABI 3730XL . The primer of TA cloning sequencing was as follows: M13F-47:5′-CGCCAGGGTTTTCCCAGTCACGAC-3′. The primer designed with Primer Premier 5.0 was used and showed as follows: forward primer, 5′-AAGGCTATTAGAAAGCAGAC-3′; reverse primer 5′-ATATGACCCAGAAACAAGAA-3′.The variant of Tissues were homogenized on ice in the lysis buffer. After centrifugation and protein quantification, proteins were loaded onto SDS–PAGE gels and transferred onto nitrocellulose membranes. Membranes were incubated in 5% nonfat dry milk in TBST for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies. Rabbit anti-DSC2 and mouse anti-β-actin antibody were purchased from Abcam Inc . Antibodies were detected using 1:10,000 horseradish peroxidase-conjugated, donkey anti-rabbit, and donkey anti-mouse IgG . The Western blot luminol reagent was used to visualize bands corresponding to each antibody.4–V6  suffered from hypertension and stroke. He was persistently laid in bed for three years and subsequently died at the age of 67 years. I: 2 (mother of II: 1) died of SCD induced by acute myocardial infarction on the way to the hospital at 73 years old. We could not receive clinical information and blood samples of I: 1 and I: 2. The echocardiograms of II: 2 and II: 3 were normal. No characteristic change related to CMs was observed in the ECGs of II: 2 and II: 3  and DSC2 p.I520T (NM_004949: EX11/CDS11: c.1559T>C), which were located in chromosome 18. The local and 1000 Genomes Project database frequency of DSC2 p.K47Rfs*2 and p.I520T was less than 0.001. DSC2 p.K47Rfs*2 was not detected in gnomAD exomes combined population. Desmocollin2 encoded by the DSC2 gene was an important component for desmosome assembly. Abnormal desmocollin2 caused the dysfunction of cell–cell adhesions and intercellular gap junctions, failing to hold together the cardiomyocytes, fibrofatty myocardial replacement, cardiac conduction delay, mechanically ventricular dysfunction and arrhythmias [DSC2 p.K47Rfs*2 converted the 47th amino acid (AA) from lysine to arginine and led to a premature termination codon at the 48th AA, which was therefore considered as frameshift and truncated mutation. This phenomenon caused the interrupting protein synthesis at the cadherin pro domain and loss of protein structure, including extracellular domains 1–4, extracellular anchor, a transmembrane domain, intracellular anchor, intracellular cadherin-like sequence, resulting in truncated protein without normal function  was demonstrated to be benign or likely benign by Clinvar database and the CHEO Genetics Diagnostic Laboratory in Children’s Hospital of Eastern Ontario (https://www.ncbi.nlm.nih.gov/clinvar/variation/155783/). The MAF of DSC2 p.I520T was 0.02% and not fulfilled with the criteria of the vast majority of reported pathogenic variants (MAF < 0.01%). Additionally, according to the analysis of the UCSC Genome Browser, the amino acid site of DSC2 p.I520 is not conserved in several species  by target capture sequencing, and the rest of the family members were subjected to Sanger sequencing. The genetic screening revealed that the proband (II: 1) carried 10 exonic variants Table , includihythmias . Accordi by targeMYH7, MYBPC3, TPM1, TAZ, TTN, and NONO genes were the most common genes causing LVNC. LVNC caused by MYH7 and MYBPC3 mutations often complicated congenital heart diseases (CHD), such as atrial septal defect, ventricular septal defect, Ebstein, tetralogy of Fallot, patent ductus arteriosus, patent foramen ovale, and aortic hypoplasia. Some cases occurred with thromboembolism. Recently, LVNC induced by DES and DSP mutations was common and young-onset with HCM, DCM, ACM, myocarditis and HF, complicating ventricular and atrial tachycardias, and even needed therapy of heart transplantation in some cases. LVNC was associated with FBN1 mutation combined with DCM and Marfan syndrome. LVNC induced by HCN4 and EMD mutations complicated sick sinus syndrome (SSS), AF, atrial standstill, and interventricular block. Additionally, the LVNC induced by EMD mutation was combined with DCM. The LVNC caused by RYR2, KCNQ1, and KCNH2 mutations occurred with catecholamine-sensitive VT, long QT syndrome (LQTs), torsade de pointes, and ventricular fibrillation. In contrast, the LVNC result from SCN5A mutation complicated SSS, AF, LQTs, Wolff–Parkinson–White syndrome, VT, and atrioventricular block. Some genes led to LVNC and caused complex and critical clinical disease syndromes, such as Rubinstein–Taybi syndrome , Ehlers–Danlos syndrome , Emery–Dreifuss muscular dystrophy , Danon disease , intellectual disability syndrome , Sotos syndrome , LEOPARD syndrome , Coffin-Lowry syndrome , Cornelia de Lange syndrome , Barth syndrome , and Holt–Oram syndrome . Additionally, ARFGEF2, MIPEP, NONO, SH2B1, and TMEM70 led to LVNC complicating developmental delay. The deletion of one of these genes, including FKBP12, JARID2, NUMB, and PLZND1, induced LVNC by affecting the activity of the Notch signaling pathway in animal models, which had not been discovered in clinical cases until now. For these genes from Table ACTC1, ACTN2, DTNA, LDB3, MIB1, MYBPC3, MYH7, PRDM16, TNNT2, and TPM1 were reported to be associated with LVNC in the OMIM database  of MYH7, MYBPC3, TPM1, TAZ, TTN, and NONO genes are the most common pathogenic mutations in LVNC [th exon deletion of KCNQ1 induced mild Beckwith-Wiedemann and severe LQTs [LVNC is a rare primary CM and serves as a global cardiac disease induced by the arrest of normal embryogenesis of the endocardium and mesocardium that leads to prominent trabeculations and deep intertrabecular recesses within the LV wall communicating with the cavity, a thin compacted epicardial layer, and acquired ventricular wall remodeling . The inf in LVNC , 32, 33, in LVNC . These c in LVNC . Some LV in LVNC . Therefo in LVNC . Actuallere LQTs . NotablyFKBP12-deficient mouse model for LVNC [FKBP12-deficient hearts. The planar cell polarity (PCP) signaling is also an essential molecular mechanism of polarity establishment for epithelial cells in planes orthogonal to the apical-basal axis. The noncanonical Wnt signaling, which is independent of β-catenin, is critical for PCP signaling. Together, they serve as the Wnt/PCP signaling pathway, which is composed of core components , PCP regulator  and a large number of PCP effectors  [Two independent biological events leading to abnormal trabeculations and compaction, including hypertrabeculation and noncompaction, are demonstrated by the for LVNC , 39. Hypfor LVNC . The enhfor LVNC , 42. Thend PhoA) , 44. Thend PhoA) .DSC2 p.K47Rfs*2 is a truncated and loss-of-function mutation, likely to cause LVNC and HCM. Compared to the MAF (< 0.01%) of inherited arrhythmia and CMs in the population, the MAF of DSC2 p.I520T (0.02%) is relatively high. Moreover, DSC2 p.I520T is not conservative in multiple species. In addition, the ECG is the main tool to judge the early changes of CMs. Especially, the abnormal changes of ECG caused by CMs are often earlier than that of the cardiac structure illustrated by echocardiography [DSC2 p.I520T and without cardiac event showed normal ECG and echocardiography. Therefore, we speculated that DSC2 p.I520T might not be pathogenic. In fact, as an important component of the cardiac desmosome, desmocollin2 maintains the normal electromechanical connection among cardiomyocytes. The desmocollin2 abnormality would lead to various CMs (such as DCM and HCM), HF, complex arrhythmia and even SCD. Recent research and our literature review showed that the abnormal components of the cardiac desmosome, including DES and DSP genes, are associated with LVNC. Desmosome is the intracellular structure that anchors intermediate filament to the plasma membrane. Desmosome, adherent junction, and gap junction are the major components of the intercalated disc, which is vital for the cell–cell adhesion and electronic coupling of cardiomyocytes [DSG2) are the important familial members of cadherin. Desmocollin2 and desmoglein2 form the adhesive heterodimer, which provides a structural framework for desmosome assembly together with armadillo proteins  and the plakin family  [DES), also expressed in cardiac desmosomes, connects Z-bands, costameres, and nuclei with the cytoskeleton. The pathogenic mutations associated with desmosome proteins may cause desmosome dysfunction and remodel intercalated disc, leading to the disturbance of the mechanical–electrical coupling of cell–cell adhesion and further alteration of signaling pathways.In this study, the proband (II: 1) carried DSC2 p.K47Rfs*2 and p.I520T variants. According to the ACMG classification criteria, the iography . Whereasmyocytes . Desmocoencodes) . The desPKP2 lead to LVNC and rapid HF, whereas the heterozygous DES p.A337P  induces LVNC and skeletal myopathy [DSC2 variant  in a patient with sporadic LVNC, but whether the variant is the cause of LVNC remains unclear [DSC2 deletion in zebrafish induces the reduction of the desmosome plaque area, deficiency of desmosome extracellular electron-dense midlines, and disturbance of myocardial contractility [DSC2 p.Q554X (c.1660C>T) participates in the pathogenicity of familial ACM [DSC2 p.A897fs*900 shows an obvious reduction in desmosome binding to plakoglobin and plakophilin [DSC2 (c.2553delA) decreases the connexin 43 expression and lost plakoglobin signal [DSC2 activates the AKT/β-catenin pathway. AKT inhibition suppresses β-catenin-dependent transcription and proliferation, which are caused by DSC2 knockdown [DSC2 protein sequence at the cadherin pro is replaced by arginine, leading to premature codon termination. Consequently, DSC2 p.K47Rfs*2 remarkably and abnormally reduced the functional desmocollin2 expression, which may interfere with desmosome formation and the stability of the intermediated disc, and disturb the mechanical stress and electrical signal propagation of cardiomyocytes. Notably, the pathological and imaging changes induced by DSC2 p.K47Rfs*2 are observed in LV, resulting in the subendocardial thickening of the anterior lateral wall and interventricular septum, and thinning in part of the left ventricular wall. The ECG of the proband showed low voltage in the limb leads but no typical ST-T change, commonly suggesting diffuse myocardial lesion. The VT arises from the middle-posterior septum and lateral wall of LV. PVCs originate from the lateral wall and apex of LV and the left ventricular inflow tract, and this finding is consistent with the cardiac lesion extent. Additionally, trabeculae are observed with serious dysplasia and distributed in a rough and disordered pattern. An apparent slow blood flow is observed in the recess of trabeculae, which may increase the risk of thrombus. However, the mechanism of DSC2 p.K47Rfs*2 that induces overlap phenotypes of LVNC and HCM remains unknown. There is no research about the relationship among DSC2, Notch, and Wnt/PCP signaling pathways.Previous studies show that the homozygous deletion mutation of myopathy , 50. A r unclear . A previactility . Additiolial ACM . DSC2 p.kophilin . The truDSC2 p.K47Rfs*2 leading to abnormalities of the desmosome structure and function should be further explored. Second, DSC2 p.K47Rfs*2 is the main cause of LVNC and HCM but still cannot rule out DSC2 p.I520T, or other variants may increase the risk of LVNC and HCM phenotype penetrance. Third, the mechanism of how the DSC2 p.K47Rfs*2 induces overlap phenotypes needs further research. Fourth, the parents and grandparents of the patient (II: 1) could not be tracked because of death. The II: 1, II: 2 and II: 3 members declined to to carry out clinical examinations and genetic testing for their offspring. Therefore, long-term follow-up is needed in the future.The limitations of the present study are as follows. First, the molecular mechanism of DSC2 p.K47Rfs*2 mutation remarkably and abnormally reduced the functional desmocollin2 expression, which may consequently induce the overlap phenotypes of LVNC and HCM, complicating AF, VT, and HF. The LVNC may be one of the important phenotypes for DSC2 pathogenic mutations.The desmocollin2 was an important component of cardiac desmosome assembly, of which abnormality caused the dysfunction of cell–cell adhesions and intercellular gap junctions. The novel heterozygous


Maternal nutrition is closely linked to the survival and development of children during the first 1000 days of life. Maternal wasting, a measure of malnutrition, is measured using the mid-upper arm circumference. However, in 2019, the rate and distribution of wasting among pregnant and lactating women was not known. We described annual trends and distribution of wasting among pregnant and lactating women (PLW), Uganda, 2015–2018, to inform programming on targeted nutritional interventions.We analyzed nutrition surveillance data from the District Health Information System for all PLW from 2015 to 2018. We used the World Health Organization standard thresholds to determine wasting among PLW by year and region, drawing choropleth maps to demonstrate the geographic distribution of wasting among PLW. We used logistic regression to assess wasting trends.p < 0.001). Regions in the north had increasing trends of wasting over the period [Lango  and Acholi ], as did regions in the east . The other 11 regions showed declines.During 2015–2018, 268,636 PLW were wasted . Of the 15 regions of Uganda, Karamoja  and Lango  registered the highest prevalence while Toro  and Kigezi  registered the lowest prevalence. The national annual prevalence of wasting among PLW declined by 31% from 2015 to 2018 (OR = 0.69, The trend of wasting among PLW nationally declined during the study period. Lango and Acholi regions, both of which were experiencing a nutrition state of emergency during this period, had both high and rising rates of wasting, as did the Karamoja region, which experienced the highest wasting rates. We recommended that the Ministry of Health increases its focus on nutrition monitoring for PLW and conduct an analysis to clearly identify the factors underlying malnutrition specific for PLW in these regions. Malnutrition refers to deficiencies, excesses, or imbalances in a person’s intake of energy and/or nutrients. It includes both undernutrition, which covers stunting, wasting, and micronutrient deficiencies, and overnutrition . WastingAdequate nutrition is crucial for the survival, health, and development of mothers and their children , 5. DuriDespite a decline in the prevalence of malnutrition globally, undernutrition remains a challenge of public health concern worldwide. In 2016, the global prevalence of underweight (9.7%) and anemia (32.8%) among women of reproductive age (15–49 years) were still above the World Health Assembly (WHA) 2025 targets of 5% for underweight and 15% for anemia –12. In UIn response to the National Development Plan 2010–2015) objective of improved nutrition in the Ugandan population, Uganda began scaling up the Nutrition Strategy with a focus on the first 1000 days of life, which refers to the period of time (for both the mother and the fetus/infant) from conception to 2 years of age  objectiv. In 2011During the 5 years of implementing the strategy (2011–2016), the prevalence of thinness (wasting) among non-pregnant or lactating women of reproductive age in Uganda declined from 11 to 9%. During the same period, wasting among children also declined from 5 to 4% and stunting declined from 33 to 29% . HoweverOur study utilized data collected from all the 15 subregions of Uganda, with 135 districts , 18. In We conducted a retrospective descriptive analysis of nutrition surveillance data from the District Health Information Software 2 (DHIS2), which are submitted quarterly from all the health facilities in Uganda. Our study focused on wasting because it accounts for higher proportions of intrauterine growth retardation (IUGR) and impaired fetal development, which carry lifelong adverse effects in children , 20.As per the Uganda Ministry of Health (MoH), a case of wasting in a PLW is defined as a mid-upper arm circumference (MUAC) in the red (< 19.0 cm) or yellow (≥19.0 to < 22.0 cm) zone on the MUAC tape . The MoHWe extracted data from DHIS2 from 2015 to 2018. The variables obtained were PLW assessed for wasting using MUAC, PLW with yellow MUAC, and PLW with red MUAC. We also extracted data on the reporting rates of the HMIS 106a form for the same period to estimate rates of under-reporting.We extracted nutrition data from DHIS directly into Microsoft Excel for cleaning. We excluded all districts with more malnourished PLW than those seen at ANC from the analysis. We imported the data into Epi-Info version 7 for analysis. We calculated the prevalence of wasting (moderate and severe) at national and regional levels, disaggregated by year. The numerator was all wasted PLW as measured by MUAC. The number of PLW assessed for wasting using MUAC within the analysis period was used as the denominator to calculate prevalence. We used the WHO categorization of wasting by prevalence which considers< 5% acceptable, 5–9% poor, 10–14% serious and ≥ 15% critical to guide interventions , 23.. ThOf the 4,848,873 pregnant and lactating women assessed for wasting over the study period, 268,636 (5.5%) were wasted. The median prevalence was 6.8%. Regions in the northern part of the country had the highest overall prevalence of wasting, while those in the regions in the western part had the lowest overall prevalence Fig. .Fig. 1PAccording to the WHO classification of malnutrition, Karamoja and Lango were in the critical malnutrition category  while Acholi was in the serious malnutrition category  , Lango , Bukedi , Busoga , and Acholi  regions, while the rest of the regions had declining trend , implemented in 2015 in 15 districts of Uganda , have beDespite the overall decline in the prevalence of wasting countrywide, disaggregated data showed that some regions, especially those in the north, still have high levels of wasting among PLW. Specifically, the northern regions of Karamoja, Lango, and Acholi were found to have the highest prevalence of wasting in this evaluation. These regions have long had prolonged droughts, insecurity, livestock diseases, and flooding which have crippled crop and livestock production ; in addiDespite having the highest prevalence of wasting in our evaluation, the Karamoja region did register a decline, possibly due to the ongoing food aid it has received for over 40 years , 30. WhiBugisu region, located in eastern Uganda, showed an increase in wasting during the evaluation period, reaching the ‘serious’ severity level in 2018. This region is subject to recurrent natural disasters, such as mudslides, and floods, that cause crop loss; the most recent of these events occurred during December 2019 –36. ThesIt is likely that some PLW were counted more than once due to repeat visits to facilities; this may have led to an overestimation of the burden of wasting in our evaluation. Alternatively, the low HMIS reporting rates, especially in 2018, may have led to an underestimation of wasting.The national trend of wasting among pregnant and lactating women declined during 2015–2018. Lango and the Acholi regions had high and rising rates of wasting. Karamoja and Lango regions experienced critical malnutrition, while Acholi region experienced serious malnutrition. We recommended that the Ministry of Health sustains interventions in place for malnutrition with special attention to Karamoja, Lango, and Acholi regions. A causal analysis to clearly understand the factors underlying malnutrition among PLW in these regions should be conducted and consideration given to implementing alternative approaches to addressing malnutrition in locations with chronically high rates.


We apply these three algorithms to 39 years of historical time-series data sets, which include 11 atmospheric variables from ERA-5 reanalysis product and passive microwave satellite retrieved sea ice extent. By comparing the causality graph results of these approaches with what we summarized from the literature, it shows that the static graphs produced by NOTEARS and DAG-GNN are relatively reasonable. The results from NOTEARS indicate that relative humidity and precipitation dominate sea ice changes among all variables, while the results from DAG-GNN suggest that the horizontal and meridional wind are more important for driving sea ice variations. However, both approaches produce some unrealistic cause-effect relationships. Additionally, these three methods cannot well detect the delayed impact of one variable on another in the Arctic. It also turns out that the results are rather sensitive to the choice of hyperparameters of the three methods. As a pioneer study, this work paves the way to disentangle the complex causal relationships in the Earth system, by taking the advantage of cutting-edge Artificial Intelligence technologies.The Arctic sea ice has retreated rapidly in the past few decades, which is believed to be driven by various dynamic and thermodynamic processes in the atmosphere. The newly open water resulted from sea ice decline in turn exerts large influence on the atmosphere. Therefore, this study aims to investigate the causality between multiple atmospheric processes and sea ice variations using three distinct data-driven causality approaches that have been proposed recently: Temporal Causality Discovery Framework Non-combinatorial Optimization  Warming in the Arctic has been much faster than in the rest of the world in both observations and model simulations, a phenomenon known as the Arctic amplification . DeclineThe traditional way to discover causal relations is to manipulate the value of a variable by using interventions or real-life experiments. All other influencing factors of the target variable can be held fixed, to test whether a manipulation of a potential cause changes the target variable . SpecifiThus, the overarching goal of this study is to investigate the causality between multiple atmospheric processes and sea ice variations from sub-seasonal to seasonal timescales using data-driven causality approaches. Instead of performing multiple climate model simulations, here we focus solely on an observational-type analysis. Specifically, three distinct data-driven causality approaches, Temporal Causality Discovery Framework (TCDF) , Non-comThis paper is structured in the following sections. a − d are well-known atmospheric processes, including cloud microphysics, thermodynamics, radiation, climate dynamics, which have been studied over the past few decades. The processes e − i are summarized from more recent publications, which are still under investigation by climate scientists. We will explain processes e − i in details in the next paragraph.Due to the two-way interactions between the atmosphere and sea ice, studying causality between them is a challenging but important task, which makes it an area of high interest within polar climate community. The sea ice variations can be caused by different dynamical and thermodynamical processes. Important dynamical processes include anomalous surface wind , regionae represents the two-way effect between sea ice and net longwave flux at the surface. Based on global reanalysis , while sea ice modulates precipitation mainly through sensible plus latent heat flux .The arrow analysis , surfaceanalysis  and sateanalysis , as wellanalysis , the dowanalysis . Positivanalysis . The seaanalysis . Once thanalysis . The feesis f in  and satesis f in . The arrn summer , winter n summer  and sprin summer  producesn summer . Moreoven summer  and summn summer  have pren summer . On the n summer . The incn summer . These cn summer , reanalyn summer . In addin summer . Earlierrvations  and modervations  during moperties . The phaoperties .The receoperties . These ccampaign , global campaign . The higcampaign . TherefoAmong these studies, very few of them have demonstrated the delayed impact of one variable on another. Specifically, the net shortwave flux at the surface in early summer (May–July) is found to enhance sea ice melt with a lag of 1–4 months . MoreoveNote that most of studies mentioned above determine the changes in one variable, happening before another one, by applying time series analysis and/or composite analysis based on observations, reanalysis or model output . Among t% during the winter, when the sea ice is relatively thick and the sea ice concentration is high. During the summer, the uncertainty increases to ±15% when the melt ponds are present ’s Integrated Forecast System (IFS), with 137 hybrid sigma/pressure (model) levels in the vertical, with the top level at 0.01 hPa . The ERAAll monthly gridded data during 1980–2018 have been averaged over the Arctic north of 60°N  using armt indicates the trend component, while the st represents seasonality component. The time series has been detrended by subtracting the line of best fit from the time-series mt, where the line of best fit was obtained from a linear regression model with the time steps as the predictor. To deseasonalize the time-series, we used averaged seasonal index st to seasonally adjust the data. The seasonal index were calculated from moving averages with a 12-months seasonal window in this study , such as a Long Short Term Memory (LSTM), for time-series data. The advantage is that RNNs typically have a vanishing gradient problem: long-term information has to sequentially travel through all the cells before getting to the present processing cell and typically stalls the learning processes, sometimes even preventing any further improvements gained with learning with more data . This isW is the weighted adjacency matrix of the underlying causality graph G(W), that is j → i in G(W) if and only if Wji ≠ 0, and the random variables Ni are independent noise variables. Given n independent and identically distributed (i.i.d.) observations of the variables X1, … , Xd, written as matrix X ∈n×d, a standard estimator for W is the (regularized) least-squares estimatorλ ≥ 0 is the regularization parameter. This estimator is theoretically well-studied and satisfies desirable properties such as consistency . Concretely, the NOTEARS algorithm applies the augmented Lagrangian method . Multilayer Perceptrons are feed-forward Artificial Neural Networks with multiple hidden layers. They are trained through stochastic gradient descent and backpropagation and their function (f2 and f3 in our case) corresponds to the relation between the input and output variables.DAG-GNN  can be tX and MLP has one hidden layer of 64 neurons. Further, DAG-GNN minimizes the following loss function.A, and parameters θ for Variational Autoencoder (VAE). Further, ELBO is the Evidence Lower Bound of the VAE adopted from (h(A) is used to solve the augmented Lagrangian as done in  done in .So DAG-GNN is a more robust, non-linear model which can learn more complex relationships than NOTEARS. Having an MLP as a backbone it is capable of learning from a large training set, and giving accurate results. Furthermore, autoencoders have been proven to be useful for unsupervised learning and feature extraction. This makes DAG-GNN capable for understanding causal relationships between atmospheric variables.static model, we treat the observations of the variables summarized in temporal model, we first augment the data set by adding lagged versions of each variable, that is for each variable X we additionally consider variables X1, X2, … , X12, where Xk is a version of the variable X that is measured with a lag of k time units (in our case: months) compared to X. Here, the maximum time lag we consider is 12 months since we want to focus on causal links from sub-seasonal to seasonal timescales. We then treat the observations of the various variables at different points in time as i.i.d. observations and run NOTEARS and DAG-GNN, respectively. The graphs produced by these methods, using the augmented data, are assumed to encode the time delay of causal relations. However, in order to obtain a causal graph on the variables of X and Y in the reduced temporal graph whenever any of the variables X, X1, … , X12 is connected to any of Y, Y1, … , Y12 in the full temporal graph. The reduced temporal graph is the output of the temporal model.While TCDF requires time series data as input and explicitly models time delay of causal relations, NOTEARS and DAG-GNN assume to be provided i.i.d. observations of the variables. Similarly to other causal discovery studies in climate research , we applIn this section we present some results of the three causal discovery algorithms introduced in A, B ∈{0,1}m×m be the adjacency matrices of two unweighted graphs GA, GB on m vertices. The normalized Hamming distance between GA and GB is given by GA and GB is zero if and only if GA and GB coincide, and it is at most one . In the following, for each of the three causal discovery algorithms introduced in In this section, we treat all graphs as unweighted graphs. The normalized Hamming distance is a widely used metric to compare two unweighted graphs on the same set of vertices . Let A, kernel = 4 and kernel = 6 for layer = 1. The kernel size is related to how much the TCDF method lags the variables for the causality study. The default setting for the hyperparameters as shown with ♣ in Since the TCDF focuses on time series, only the temporal graph that is closest to the domain knowledge graph is shown . In geneλ ≥ 0 and t ≥ 0 as explained in λ is the regularization parameter .In In the static graph, the RH and precipitation seem to dominate the sea ice changes, with weights of 0.55 and 0.41, respectively. In the meantime, the sea ice exerts large influence on SW (weight of 0.79) and CW (weight of 0.31). The causal relations between precipitation, SW and sea ice in the domain knowledge graph of τ ≥ 0 and t ≥ 0, where τ is similar to λ used in NOTEARS. We noticed that the hyperparameter τ is very sensitive, and show the outputs for only two values of τ i.e., τ ∈{0, e − 7}. We vary t similar to NOTEARS, and test for t ∈{0.2, 0.3}. t.Like the NOTEARS algorithm, DAG-GNN has two hyperparameters: τ = e − 7, t = 0.3 for the static model and τ ∈{0, e − 7}, t = 0.3 for the temporal model. Both the temporal models of t = 0.3 give the same graphs, which is shown in τ and t produce a graph which shows no relation between sea ice and other atmospheric variables. Hence, we reject it. The second most optimum graph showing a dependence of sea ice with atmospheric variables is shown in τ = 0 and t = 0.2. Further, we note that for the temporal model, the best graphs are obtained with t = 0.3, which is one of the default parameters used and suggested by the authors in . In The Arctic has undergone dramatic changes in the past few decades, and sea ice decline is believed to be a key driver for the Arctic amplification. On the one hand, the sea ice is melted by mixed effects of atmospheric dynamical and thermodynamical processes. These processes on the other hand, can be largely affected by sea ice melt. Therefore, this study investigates the causality between multiple atmospheric processes and sea ice variations using three data-driven causality discovery approaches . As shown in previous sections, one advantage of utilizing these approaches is that they not only generate causal graphs, but also provide quantified information on causal strength through weights or time lags. Another advantage is that these approaches can take all relevant variables into consideration and find potentially important causal relationships, which is different from most related studies which only analyze pair-wise causality between two variables. Instead of performing computationally expensive climate model simulations, here we focus solely on an observational-based analysis. Specifically, we examine the sensitivity of causality graphs produced by three methods to different hyperparameters and then compare those graphs with domain knowledge graph.We found that the outputs of the three algorithms are rather sensitive to the choice of hyperparameters. For example, choosing an only slightly too large regularization parameter can result in NOTEARS or DAG-GNN producing empty graphs, that is not discovering any causal relationships at all. Also the values of the other parameters turned out to be important and outputs for different choices of the hyperparameters can be quite different. Hence, some care must be taken when applying data-driven causality discovery approaches and domain knowledge is indispensable for assessing whether their produced outputs are reasonable.Compared to domain knowledge graph, the static graphs produced by NOTEARS and DAG-GNN are relatively reasonable. The results from NOTEARS suggest that RH and precipitation dominate sea ice changes among all variables. In the meantime, the sea ice has a large impact on SW and CW. The graph generated by DAG-GNN, however, indicates that the zonal (u10 m) and meridional (v10 m) wind fields are more important for driving sea ice variations than other variables. And there are no atmospheric variables being affected by the sea ice. Note that the edges between u10 m and v10 m, SW and v10 m, CW and v10 m are produced by both NOTEARS and DAG-GNN, which are different from domain knowledge graph, possibly due to the averages over a large domain. As for the temporal graphs, very few edges can be found in TCDF and NOTEARS. In comparison, the DAG-GNN is able to produce more complicated and meaningful results. The sea ice is found to have a delayed impact on itself, but with no causal relationship with any atmospheric processes. This is possibly because sea ice anomaly persistence is much stronger than the connections between sea ice and atmosphere. It is our hope that those causality graphs can be compared with the ones produced by other algorithms as Artificial Intelligence technologies are evolving rapidly. In the meantime, they can be also compared with the causal links captured by physical models for cross-validation.Based on our analysis, it is still very challenging to directly apply these state-of-the-art data-driven causality discovery approaches to this specific climate topic. However, there are several limitations with current study, which potentially has a large influence on our results. 1) There are large uncertainties in the domain knowledge graph and thus cannot be considered as ground truth. Climate scientists strive to investigate the complex feedbacks between atmosphere and sea ice, but our knowledge in this field is still very limited and controversial. For example, a few recent studies have divergent consensuses on Arctic amplification’s influence on mid-latitude severe winter weather . 2) We aNevertheless, this is a pioneer study in the application of data-drive causality discovery approaches in the interactions between atmosphere and sea ice. This study will pave the way for us to disentangle the complicated causal relationships in the Earth system, by taking the advantage of cutting-edge data science and Artificial Intelligence technologies. It also provides a good opportunity for climate scientists, data scientists and computer scientists to work together to solve the puzzle in the nature, which will eventually advance our understanding of polar climate system and global climate change.


Mast cells (MCs) are an important treatment target for high-affinity IgE Fc receptor (FcεRI)-mediated allergic diseases. The plant-derived molecule 4-methylumbelliferone (4-MU) has beneficial effects in animal models of inflammation and autoimmunity diseases. The aim of this study was to examine 4-MU effects on MC activation and probe the underlying molecular mechanism(s). We sensitized rat basophilic leukemia cells (RBLs) and mouse bone marrow-derived mast cells (BMMCs) with anti-dinitrophenol (DNP) immunoglobulin (Ig)E antibodies, stimulated them with exposure to DNP-human serum albumin (HSA), and then treated stimulated cells with 4-MU. Signaling-protein expression was determined by immunoblotting. In vivo allergic responses were examined in IgE-mediated passive cutaneous anaphylaxis (PCA) and ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) mouse models. 4-MU inhibited β-hexosaminidase activity and histamine release dose-dependently in FcεRI-activated RBLs and BMMCs. Additionally, 4-MU reduced cytomorphological elongation and F-actin reorganization while down-regulating IgE/Ag-induced phosphorylation of SYK, NF-κB p65, ERK1/2, p38, and JNK. Moreover, 4-MU attenuated the PCA allergic reaction . Similarly, we found that 4-MU decreased body temperature, serum histamine, and IL4 secretion in OVA-challenged ASA model mice. In conclusion, 4-MU had a suppressing effect on MC activation both in vitro and in vivo and thus may represent a new strategy for treating IgE-mediated allergic conditions. Mast cells (MCs) are key effector cells in IgE-mediated allergic and inflammatory reactions, including asthma, allergic rhinitis, and atopic dermatitis ,2. AllerUmbelliferae and Asteraceae plant species, has anti-inflammatory effects , anti-p-SYK [1:1000], anti-ERK [1:1000], anti-p-ERK [1:1000], anti-JNK [1:1000], anti-p-JNK [1:1000], anti-p38 [1:1000], anti-p-p38 [1:1000], anti-p-p65 [1:200], anti-p65 [1:5000], anti-IκBα [1:2000] and anti-GAPDH [1:2000] primary antibodies in TBS-T. The membranes were then incubated for 1 h with HRP-conjugated secondary antibodies [1:2000]. Immunoreactive protein bands were visualized by reaction with chemiluminescent reagents . The relative levels of p-SYK/SYK, p-ERK/ERK, p-JNK/JNK, p-p38/p38, p-p65/p65 and IκBα/GAPDH were analyzed statistically using Image J software .w/v in 1% saline, pH 2.5) as described in detail elsewhere [We stained RBLs with toluidine blue  was injected intradermally into mouse ears. Then, 24 h later, topical 4-MU (50 mg/kg) was applied to ears or ketotifen  was injected intraperitoneally (i.p.) (N = 5/group). An hour later, the mice were challenged with DNP-HSA . The mice were euthanized 1 h later. The ear thickness was measured by electronic digital caliper . The ears were removed and submerged in formamide (700 μL) at 65 °C for 12 h to allow dye extraction. Dye intensity was quantitated at 620 nm by a microplate reader . Mouse ears were submerged in 4% formaldehyde and then embedded in paraffin in preparation for slicing. Finally, sections were submitted to toluidine blue staining .Mice (N = 5/group) were sensitized with OVA mixture solution  on days 0 and 7, as we have described previously . 4-MU (5p < 0.05.The data are expressed means from three independent experiments with standard deviations (SDs). One-way analyses of variance (ANOVAs) were conducted in Prism 8  with a significance criterion of IL1β, IL4, and TNFα transcription in IgE-activated RBLs following 4-MU treatment (Cytotoxic screening confirmed that 4-MU (0.1–100 μM) does not reduce RBL viability significantly b. Thus, reatment e–g.IgE-activated RBLs were identified metachromatically by the dominant presence of distinctive secretory granules in the cytoplasm. Non-activated RBLs were elongated and contained purple-colored intra-cellular particles. IgE-stimulated RBLs had irregular shapes with less staining of secretory granules in the cytoplasm due to extensive degranulation. 4-MU inhibited these changes and FcεRI-signaling induced particle release in activated RBLs a,b. AddiBecause crosslinking of Ag-bound IgE and FcεRI leads to MC degranulation via activation of the SYK, mitogen-activated protein kinase (MAPK), and nuclear factor-kappa B (NF-κB) signaling, we investigated the effects of 4-MU on SYK, MAPK, and NF-κB pathway activation in RBLs. Western blot analysis showed that pretreatment with 4-MU attenuated the phosphorylation of SYK, p38, ERK, JNK, and NF-κB p65 and increased IκBα levels in FcεRI-activated RBLs dose-dependently a–c. SimiIL1β, IL4, and IL6) expression of BMMCs  in corneal fibroblasts , LPS-indFcer1A, Ms4a2, and Fcer1G (Previously, SYK, MAPK, and NF-κB pathways have been suggested to be involved in mediating signaling downstream of FcεRI activation that promotes MC degranulation ,40. Mechd Fcer1G , suggestIt has not been clarified how or whether 4-MU inhibitory effects on HA synthesis relate to the anti-inflammatory actions 4-MU. Moreover, the molecular mechanisms of HA regulation in FcεRI-mediated MC degranulation have yet to be substantively clarified. Interestingly, our findings suggest that HA production may be associated with FcεRI-mediated activation of MCs. If so, HA synthetase could represent an important target for MC activation.4-MU is sold over-the-counter as a spasmolytic in several European countries . ExploraA typical adult 4-MU dosage range is a total of 900–2400 mg/day, divided into three daily doses . PreviouIn summary, we found that the FDA-approved plant-derived HA synthesis inhibitor 4-MU attenuates FcεRI-mediated activation of MCs and MC inflammatory responses. Our data suggest that the effects of 4-MU on inflammatory responses may involve MAPK-, SYK-, and/or NF-κB-dependent mechanisms. The presently demonstrated in vitro and in vivo effects of 4-MU on MC activation suggest that it should be considered a candidate of interest for being re-purposed as an MC inhibitor for allergic disease treatment.