The results showed that the IC50 of GLLGY, HWP, and VYGF for ACE inhibition was 1 mg/mL, and the IC50 of HWP for pancreatic lipase was 3.95 mg/mL. Molecular docking revealed that the binding energies between GLLGY, HWP, and VYGF and ACE were –9.0, –8.4, and –9.2 kcal/mol, respectively. The binding free energy between HWP and pancreatic lipase was –7.3 kcal/mol. GLLGY, HWP, and VYGF inhibited ACE compentitively. HWP inhibited pancreatic lipase through non-competition. in vitro simulated gastrointestinal digestion, the three oligopeptides still had inhibitory activity and low toxicity. The results revealed that the peptides GLLGY, HWP, and VYGF may be suitable candidates for further research on ACE inhibition, and HWP may be a suitable candidate for studying pancreatic lipase inhibition.This study determined the inhibitory activity of oligopeptides against angiotensin-converting enzyme (ACE) and pancreatic lipase through  Obesity has recently shown an epidemic trend globally . ObesityHypertension is regulated by various mechanisms, one of which is related to the activity of the angiotensin-converting enzyme (ACE) . ACE pla50 of red lentil protease hydrolysate for ACE was 111 ± 1 μmol/L  were conducted to explore whether these oligopeptides can be used in health products.In this study, The polypeptide was synthesized by Hangzhou Dangang Biotechnology Co., Ltd . Human ACE, N-[3-(2-furanyl) acryloyl]-l-phenylalanyl glycyl glycine (FAPGG), p-nitrophenyl butyrate (pNPB), and pancreatic lipase were purchased from Sigma Co., St. Louis, Missouri, United States.Bacillus subtilis MK15-fermented rice bran . The absorbance value was incorporated into the following formula to calculate the inhibition rate of polypeptide against ACE activity:According to the experimental method of Yu Fu et al. , with sl1 and b1 are initial absorbance values of the blank and sample groups, respectively. a2 and b2 are absorbance values of the blank and sample groups after the reaction, respectively. A = a1–a2; B = b1–b2.Where aAccording to the method of Magdalena mendoza-s á nchez . In the Where A, B, C, and D are absorbance values of the blank, blank control, sample, and sample blank groups, respectively.1, the crystal structures of human ACE  and porcine pancreatic lipase  were retrieved in PDB format. Subsequently, co-crystalline ligands and water molecules were removed from the protein structures using PyMOL version 2.5.0.Autodock Vina 1.1.2 docking software was used for molecular docking. From the protein databaseWhen the polypeptide was docked with ACE, the docking site was the same as that reported by Dong . The griThe inhibition kinetics of oligopeptides GLLGY, HWP, and VYGF on ACE and pancreatic lipase were tested to determine their efficiencies. For the ACE inhibition test, the concentration of the samples GLLGY, HWP, and VYGF was 1 mg/mL, and the concentrations of the substrate FAPGG were 0.2, 0.4, 0.6, 0.8, and 1 mM. For the pancreatic lipase inhibition test, the sample HWP concentration was 1 mg/mL and the substrate pNPB concentrations were 1, 2, 3, and 5 mM. The initial velocity data were used to construct the Lineweaver–Burke plots to determine the enzyme Km (Michaelis constant), Vmax (maximum velocity), and Ki (inhibitory binding constant) .in vitro pepsin–pancreatin hydrolysis method was carried out (3 solution and further to pH 7.5 with 1 M NaOH. Pancreatin was added (4% weight as received/weight of protein in the powder), and the mixture was further incubated at 37°C for 2 h. To terminate the digestion, the test tubes were kept in boiling water for 10 min. The gastric digests (GDs) and gastric-intestinal digests (GIDs) were obtained through centrifugation , and then, the supernatants were collected, lyophilized, sealed in plastic bags, and stored at 4°C until further analysis. The ACE and pancreatic lipase inhibitory activities of GDS and GID were measured at a concentration of 1 mg/mL, and the results were expressed as activity (%) relative to that without any treatment . All determinations were made in triplicate.Simulated GI digestion through an ried out . The oliin vitro evaluation of ADMET  provides data for the identification of new molecules  value represents the concentration of a molecule necessary to cause the death of 50% of the fathead minnows. LC50 values of <0.5 mM (log LC50 <-0.3) represent high acute toxicity. The LC50 values of GLLGY, HWP, and VYGF were >0.5 mM. The three oligopeptides were regarded as of low toxicity because the toxicity was likely associated with the disrupted function of the liver. These oligopeptides did not cause skin sensitization. Thus, these three oligopeptides were concluded to be of low toxicity  is considered low and that of >0.477 log (mg/kg/day) is considered high. The dose of HWP was considered low, whereas that of GLLGY and VYGF was considered high. toxicity .2+, which contain Ala354, Glu384, Tyr523, Gln281, His353, Lys511, His513, Tyr520, and Glu162 residues. GLLGY, HWP, and VYGF are docked with ACE. GLLGY formed 9 hydrogen bonds with the ACE residues Thr282, Ser284, His353, Glu376, Glu411, Gly414, Asp415, and His513 to produce 9 hydrogen bonds  are Ser153, Asp177, His264, Phe78, Ile79, His152, Phe216, Trp253, and Arg257 . Sha Li  0.03 mM . HWP int 0.03 mM ; however 0.03 mM , which m8.988 mM , which iin vitro were not considerably lower than those before digestion. Tausif Ahmed  hydrolysate (CPH) on pancreatic lipase, and found that cocoa peptides EEQR, GGER QTGVQ and VSTDVNIE had inhibitory effect on pancreatic lipase. The IC50 of CP hydrolysate was 1.38 mg/mL. In vivo experiments, compared with the high-fat diet group, the high-fat diet and CP group significantly reduced the apparent absorption rate of fat  can reduce systolic blood pressure (SBP) by about 22 mmHg and diastolic blood pressure (DBP) by about 16 mmHg in vivo (in vitro, furthermore, they had anti digestion ability. It is preliminarily inferred that they might have an inhibitory effect in vivo and promote human health. The oligopeptides extracted from fermented rice bran are worthy to be investigated the effect in vivo and application in further.Study showed that oligopeptides could inhibit pancreatic lipase and ACE e of fat . Li Peng in vivo . Our oliin vitro. The IC50 of GLLGY, HWP, and VYGF for ACE inhibition was 1 mg/mL and that of HWP for pancreatic lipase was 3.95 mg/mL. Molecular docking results showed that 9, 11, and 6 hydrogen bonds were present between GLLGY, HWP, and VYGF and ACE, respectively. Nine hydrogen bonds were present between HWP and pancreatic lipase. GLLGY, HWP, and VYGF competitively inhibited ACE, and HWP non-competitively inhibited pancreatic lipase. The oligopeptides GLLGY, HWP, and VYGF continued to exhibit strong inhibitory activity during simulated GI digestive activity. Three oligopeptides are low toxicity. The oligopeptides extracted from fermented rice bran have great prospective application for protect human health.The biological activity of better peptide components was determined The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.JH: conceptualization and writing—original draft preparation. HW: data curation and resources. NW: visualization and investigation. TW: supervision. XT: methodology. JL: software and validation. ML: writing—review and editing. SW: funding acquisition and project administration. All authors contributed to the article and approved the submitted version.


The US Food and Drug Administration (FDA) recently restricted characterizing flavors in tobacco products. As a result, ice hybrid–flavored e-cigarettes, which combine a cooling flavor with fruit or other flavors , emerged on the market. Like menthol, ice-flavored e-cigarettes produce a cooling sensory experience. It is unclear if ice hybrid–flavored e-cigarettes should be considered characterizing flavors or menthol, limiting regulatory action. Monitoring the public’s conversations about ice-flavored e-cigarettes on Twitter may help inform the tobacco control community about these products and contribute to the US FDA policy targets in the future.This study documented the themes pertaining to vaping and ice flavor–related conversations on Twitter. Our goal was to identify key conversation trends and ascertain users’ recent experiences with ice-flavored e-cigarette products.Posts containing vaping-related  and ice-related  terms were collected from Twitter’s streaming application programming interface from January 1 to July 21, 2021. After removing retweets, a random sample of posts (N=2001) was selected, with 590 posts included in the content analysis. Themes were developed through an inductive approach. Theme co-occurrence was also examined.Many of the 590 posts were marked as (or consisted of) marketing material , contained positive personal testimonials , and mentioned disposable pods . Other themes had relatively low prevalence in the sample: neutral personal testimonials , cannabidiol products , negative personal testimonials , “official” flavor description , ice-flavored JUUL , information seeking , and comparison to combustible tobacco . The most common co-occurring themes in a single tweet were related to marketing and disposable pods .Our findings offer insight into the public’s experience with and understanding of ice-flavored e-cigarette products. Ice-flavored e-cigarette products are actively marketed on Twitter, and the messages about them are positive. Public health education campaigns on the harms of flavored e-cigarettes may help to reduce positive social norms about ice-flavored products. Future studies should evaluate the relationship between exposure to personal testimonials of ice-flavored vaping products and curiosity, harm perceptions, and experimentation with these products among priority populations. The use of e-cigarettes, or vaping, among adolescents and young adults is a public health concern ,2. One fIce hybrid–flavored products may contain menthol. Menthol is a flavor additive that produces pleasant cooling sensations and analgesic effects in the throat and mouth, which reduces the harshness of combustible smoke and nicotine’s irritating effects on the airways ,12. ReseOne approach that may be helpful in informing regulatory efforts is the tracking and analyzing of real-world conversations and depictions of novel tobacco products on Twitter . MonitorThis study used Twitter data to document the themes in posts pertaining to ice flavors. Examining and monitoring the public’s conversations about ice-flavored e-cigarettes may help inform the tobacco control community about these products and uncover trends, knowledge, and attitudes toward e-cigarette flavors. Findings from this study may contribute to the US FDA policy targets and tobacco control campaigns in the future.Twitter posts containing both vaping-related  tweets that were included in the content analysis.personal testimonials , marketing, official  flavor description, disposable pod devices, JUUL, cannabidiol (CBD) products, and other. A tweet could be classified into multiple themes. To establish interrater reliability, the same subsample of posts  were double coded, with percent agreement ranging from 99.2% to 100% and Cohen κ ranging from 0.90 to 1.00. JPA served as an arbitrator resolving discrepancies between the coders.The codebook consists of the following themes: personal testimonials consisted of positive, negative, and neutral testimonies, personal testimonials theme, and only includes its components: positive, negative, and neutral testimonies. The other category was also excluded since it had few co-occurrences with other themes.Descriptive analyses were conducted first to show the prevalence of each theme. Further, pairwise co-occurrence analyses were used to evaluate connections across themes, since tweets often included more than one theme. R base package  was used to construct the co-occurrence matrix, and the igraph package (version 1.2.6) was used to visualize the results. Since the theme related to All Twitter posts in this data set were publicly available and anonymized, and all analyses adhered to the terms and conditions, terms of use, and privacy policies of Twitter. To further protect privacy, posts exemplifying themes were paraphrased; no tweets are reported verbatim.The protocol was approved by the university’s institutional review board (protocol HS-18-00697).marketing  of ice-flavored products and personal testimonials . Mentions of using ice-flavored disposable pod devices  was also a common theme. Other themes had relatively low prevalence in the sample: “ice-flavored” CBD products , official flavor description , and ice-flavored JUUL . Positive sentiment  was the most prevalent theme among personal testimonials, while the other testimonial themes were less common: neutral sentiment , negative sentiment , information seeking , and comparison to combustible tobacco .Descriptive characteristics of the 590 tweets from this corpus and example paraphrased posts are presented in marketing, positive sentiment, and disposable pod devices. The marketing of ice-flavored disposable pod devices was mentioned in 73 (12.4%) of the 590 tweets; the marketing of ice-flavored CBD products was mentioned in 40 (6.8%) of the tweets; the marketing of ice-flavored products and official flavor descriptions was mentioned in 37 (6.3%) of the tweets. Positive testimonies about disposable devices were observed in 31 (5.3%) of the tweets, and conversations about disposable devices containing CBD were observed in 26 (4.4%) of the tweets. Pairwise co-occurrences of the remaining themes were found in 2% of the tweets  and refillable  e-cigarette products . These dPersonal testimonials was another common theme in this study, while positive sentiment was the most common category among testimonials. These findings are consistent with previous studies showing that Twitter users are exposed to positive messages about vaping and tobacco [ tobacco ,26. Posi tobacco . Given t tobacco ,26. For This study was limited to the analysis of discussions on ice-flavored e-cigarette products and may not pertain to other e-cigarette flavors. In addition, relatively few  tweets among all identified ice tweets were analyzed , which limits the generalizability of our results. Moreover, the word “cool” was one of the four terms used to identify ice-flavored e-cigarette posts. “Cool” is a colloquialism and is commonly used to express the enjoyment of someone or something. The inclusion of this search term resulted in a collection of many posts that were unrelated to e-cigarette flavors; nonetheless, we found this limitation unavoidable and worked diligently to analyze pertinent tweets. This study focused on the text of the Twitter posts but did not code website links or accompanying images. Previous studies demonstrated that there is value in examining both text and image. In other words, it is possible that additional themes would have emerged had we coded images. Findings may not generalize to other time periods or other social media platforms. Our findings may not extend to all Twitter users or to the population of the United States.Our findings may offer valuable insights into the public’s experience with and understanding of ice-flavored e-cigarette products. In this study, we found that Twitter discussions about ice-flavored e-cigarette products focused on marketing, personal testimonials, and ice-flavored disposable pod devices. Future studies should evaluate the relationship between exposure to personal testimonials of ice-flavored vaping products and curiosity, harm perceptions, and experimentation with these products among priority populations. Public health education campaigns informing the public on the harms of flavored e-cigarettes may be helpful in reducing positive social norms about ice-flavored products, while flavor regulatory actions may include ice flavors to prevent new products from circumventing current regulations. For instance, banning tobacco marketing posts on Twitter may reduce the exposure to ice-flavored e-cigarette product promotions. In addition, social media might be more influential than traditional marketing because participants can be actively engaged in the content they are viewing ,22. Clos


LRRK2 gene are a common monogenic cause of Parkinson's disease. However, only seven variants have been confirmed to be pathogenic.Pathogenic variants in the LRRK2 variants (H230R and A1440P) and performed functional testing.We identified two novel We transiently expressed wild‐type, the two new variants, or two known pathogenic mutants (G2019S and R1441G) in HEK‐293 T cells, with or without LRRK2 kinase inhibitor treatment. We characterized the phosphorylation and kinase activity of the mutants by western blotting. Thermal shift assays were performed to determine the folding and stability of the LRRK2 proteins.The two variants were found in two large families and segregate with the disease. They display altered LRRK2 phosphorylation and kinase activity.LRRK2 variants which segregate with the disease. The results of functional testing lead us to propose these two variants as novel causative mutations for familial Parkinson's disease. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder SocietyWe identified two novel  Indeed, the G2019S pathogenic variant is the most frequent, with its prevalence reaching up to 29% in Ashkenazi Jewish and 37% in North African Berber populations.LRRK2 have been reported to be linked to PD thus far,Pathogenic variants in the leucine‐rich repeat kinase 2 : c.689A>G, p.(His230Arg) in exon 6 in family 1  and serine 935 , whereas the R1441G mutant showed 2.5‐fold lower phosphorylation at serine 910 (P = 0.009), as expected,P = 0.215). The H230R mutant showed a slight but nonsignificant increase in phosphorylation at serine 910 (P = 0.561) and a 2.4‐fold increase at serine 935 (P = 0.003), whereas the mutant A1440P showed 3.5‐fold decreased phosphorylation at serine 910 (P = 0.003) and a slight but nonsignificant decrease in phosphorylation at serine 935 (P = 0.319).We first studied the phosphorylation of LRRK2 by other kinases at serine 910 and 935 Fig. . In HEK‐P < 0.0001).P = 0.997). The mutant H230R showed 2.8‐fold greater phosphorylation at serine 1292 (P = 0.013), whereas A1440P showed no difference relative to WT (P = 0.999).We then studied the phosphorylation of serine 1292 Fig. , which iP = 0.029) and 4.8‐fold higher phosphorylation for R1441G (P < 0.0006). The rate of phosphorylation of RAB10 was approximately 3.1‐fold higher for H230R and 3.7‐fold higher for A1440P   Fig. .3.3P = 0.013), serine 935 for H230R (P = 0.003) and G2019S (P = 0.032), and serine 1292 for H230R (P = 0.042) and G2019S (P = 0.006). Treatment with MLi‐2 almost completely suppressed the phosphorylation of RAB10 at threonine 73 under all conditions  provide sufficient evidence to consider them to be at least “likely pathogenic” for A1440P according to the American College of Medical Genetics (ACMG) guidelines for the classification of genetic variants.We identified two novel potentially disease‐causing variants of the LRRK2 A1440P shows phosphorylation rates at serine 910, 935, and 1292 similar to those of R1441G located at the adjacent position.Limitations of our study are the use of a transient gene expression and the method of detection we used. Our study should be confirmed in other models as inducible stable expression system and/or using other quantification methods such as quantitative multiplexed immunoblot using fluorescently‐labeled secondary antibodies or measurement of LRRK2‐dependent Rab10 phosphorylation in patient‐derived peripheral blood neutrophils by immunoblotting as well as by targeted mass spectrometry.Drosophila melanogaster has identified altered pathways associated with N551K, including alterations of the oxidoreductase pathway.Given its location and the similar phosphorylation pattern of the mutated protein, the pathological effects of A1440P are likely to be similar to those of pathogenic variants located in the Roc‐COR domain, such as R1441G. However, the pathophysiology and increased kinase activity of the H230R variant, located in the ARM domain, is less obvious. Another reported variant in this domain, A211V, also showed a slight increase in kinase activity.LRRK2 variants, H230R and A1440P, which segregate with the disease in large PD families. We show that H230R and A1440P alter the phosphorylation rates of LRRK2 and its ability to phosphorylate its substrate RAB10. Further studies on these rare potentially disease‐causing variants should help us to better understand how LRRK2 dysfunction causes PD and may have implications for future treatment strategies against LRRK2‐related disorders.In conclusion, we have identified two novel The authors declare that there is no conflict of interest.This work was funded by the University of Lille, the “Institut National de la Santé et de la Recherche Médicale” (INSERM), the Lille University Hospital, the Association des Aidants et Malades à Corps de Lewy (A2MCL) charity, and the France Alzheimer charity. This work was also supported by grants from the Programs d'Investissements d'Avenir LabEx (excellence laboratory) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to ALZheimer's disease), the ANR  grant ANR‐16‐CE16‐0012‐02 MeTDePaDi, grant ANR‐20‐CE16‐0008 Synapark, grant ANR‐21‐CE16‐0003‐01 PARK‐PEP, Fondation de France , France Parkinson (R16008), and The Michael J. Fox Foundation, grant numbers 6709.03, 10255.03, and 12938.04, and the Protocole Hospitalier de Recherches Cliniques Convergence (CPP/2008/009).(1) Research Project: A. Conception, B. Organization, C. Execution, D. Data Curation; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript Preparation: A. Writing of the First Draft, B. Review and CritiqueI.C.: 1A, 1C, 1D, 2B, 3AE.M.: 1B, 1C, 1D, 3BS.E.: 1B, 1DS.C.: 1B, 1DA.M.: 1B, 1DC.D.: 1B, 1DL.G.: 1B, 1DG.B.: 1B, 1DM.T.: 1B, 3BR.M.: 1C, 1D, 3AX.T.: 1B, 3BG.V.: 1D, 3BW.V.: 1D, 3BL.B.: 1B, 3BL.D.: 1B, 3BB.S.: 1B, 3BM.‐C.C.‐H.: 1A, 1B, 2B, 3BJ.‐M.T.: 1A, 2A, 2B, 3BV.H.: 1A, 1B, 1D, 2A, 2B, 3A, 3BI.C.: None.E.M.: Protocole Hospitalier de Recherches Cliniques Convergence (CPP/2008/009).S.E.: None.S.C.: None.A.M.: None.C.D.: None.L.G.: None.G.B.: None.M.T.: None.R.M.: None.X.T.: The CANTHER laboratory “CANcer, heterogeneity, plasticity and resistance to THERapies” is supported by a grant from “Contrat de Plan Etat‐Région” Cancer 2015–2020.G.V.: None.W.V.: FWO (Research Foundation – Flanders), grants G0D5217N and G072821N.L.B.: Programs d'Investissements d'Avenir LabEx (excellence laboratory) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to ALZheimer's disease).L.D.: Board: Abbvie; Honoraires symposium: UCB, Abbvie, Orkyn.B.S.: None.M.‐C.C.‐H.: ANR  grant ANR‐16‐CE16‐0012‐02 MeTDePaDi, grant ANR‐20‐CE16‐0008 Synapark, grant ANR‐21‐CE16‐0003‐01 PARK‐PEP, Fondation de France , France Parkinson (R16008), Association des Aidants et Malades à Corps de Lewy (A2MCL) charity, France Alzheimer charity, and The Michael J. Fox Foundation, grant numbers 6709.03, 10255.03, and 12938.04, and the Protocole Hospitalier de Recherches Cliniques Convergence (CPP/2008/009).J.‐M.T.: ANR  grant ANR‐16‐CE16‐0012‐02 MeTDePaDi, grant ANR‐20‐CE16‐0008 Synapark, grant ANR‐21‐CE16‐0003‐01 PARK‐PEP, Fondation de France , France Parkinson (R16008), and The Michael J. Fox Foundation, grant numbers 6709.03, 10255.03, and 12938.04.V.H.: Association des Aidants et Malades à Corps de Lewy (A2MCL) charity, France Alzheimer charity, Programs d'Investissements d'Avenir LabEx (excellence laboratory) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to ALZheimer's disease).Appendix S1 Supporting InformationClick here for additional data file.TABLE S1 List of primer names and sequences used for site‐directed‐mutagenesisClick here for additional data file.TABLE S2 Antibodies used in immunohistochemistry experimentsClick here for additional data file.TABLE S3 Results of the mutation prediction toolsClick here for additional data file.FIG. S1Click here for additional data file.FIG. S2Click here for additional data file.FIG. S3Click here for additional data file.FIG. S4Click here for additional data file.


Fluctuations in Antarctic Ice Sheet elevation and mass occur over a variety of time scales, owing to changes in snowfall and ice flow. Here we disentangle these signals by combining 25 years of satellite radar altimeter observations and a regional climate model. From these measurements, patterns of change that are strongly associated with glaciological events emerge. While the majority of the ice sheet has remained stable, 24% of West Antarctica is now in a state of dynamical imbalance. Thinning of the Pine Island and Thwaites glacier basins reaches 122 m in places, and their rates of ice loss are now five times greater than at the start of our survey. By partitioning elevation changes into areas of snow and ice variability, we estimate that East and West Antarctica have contributed −1.1 ± 0.4 and +5.7 ± 0.8 mm to global sea level between 1992 and 2017. We combine satellite radar altimetry and a regional climate model to separate changes in Antarctic elevation into snow and ice contributionsBetween 1992 and 2017, ice thinning has grown in extent to include 24% of West Antarctica and reaches 122 m in placesIce losses from Pine Island and Thwaites Glaciers have risen fivefold; East and West Antarctica have contributed 4.6 ± 1.2 mm to sea level In other areas, where the altimeters failed to track rugged terrain (<2%) and in gaps between the satellite ground tracks, we interpolated the observed elevation changes using a triangulation scheme  the method of determining the satellite range, which has been done with a variety of waveform retracking routines , a broad pattern of modest ice sheet thickening spans much of the coastline and stretches several hundred kilometers inland; this has been associated with sharp increases in snowfall that occurred between 2009 and 2012  of the continental ice sheet for ERS‐1, ERS‐2, and ENVISAT, the broader pattern of elevation change within the Antarctic interior has remained stable over the survey period and is consistent with the changes recorded farther south by CryoSat‐2 since 2010; that is, the Kamb, Whillans, and Mercer ice streams have thickened, steadily, while little change has occurred elsewhere. Our estimate of the ice sheet interior mass balance is based on elevation trends recorded in the southernmost degree of latitude sampled and puts the region in a state close to balance—gaining 1.7 ± 0.1 Gt/year, on average, since 1992. At the continental scale, we estimate that the EAIS and WAIS changed in mass at average rates of +16 ± 6 and −82 ± 12 Gt/year, respectively, between 1992 and 2017 (Table % of the By far, the largest signal of imbalance has occurred in the Pine Island and Thwaites glacier drainage basins Figure , which hAccelerating mass loss from the Amundsen Sea Sector has been documented in a wide range of satellite altimetry (Thomas et al., 5Our ERS‐1, ERS‐2, ENVISAT, and CryoSat‐2 radar altimetry time series provides a comprehensive assessment of the spatial and temporal pattern of AIS elevation change between 1992 and 2017. When combined with model estimates of firn thickness change (Ligtenberg et al., Supporting Information S1Click here for additional data file.


Biomechanical performance of longitudinal component in dynamic hybrid devices was evaluated to display the load-transfer effects of Dynesys cord spacer or Isobar damper-joint dynamic stabilizer on junctional problem based on various disc degenerations. The dynamic component was adapted at the mildly degenerative L3–L4 segment, and the static component was fixed at the moderately degenerative L4–L5 segment under a displacement-controlled mode for the finite element study. Furthermore, an intersegmental motion behavior was analyzed experimentally on the synthetic model under a load-controlled mode. Isobar or DTO hybrid fixator could reduce stress/motion at transition segment, but compensation was affected at the cephalic adjacent segment more than the caudal one. Within the trade-off region , the stiffness-related problem was reduced mostly in flexion by a flexible Dynesys cord. In contrast, Isobar damper afforded the effect of maximal allowable displacement  to reduce stress within the pedicle and at facet joint. Pedicle-screw travel at transition level was related to the extent of disc degeneration in Isobar damper-joint (more than Dynesys cord spacer) attributing to the design effect of axial displacement and angular rotation under motion. In biomechanical characteristics relevant to clinical use, longitudinal cord/damper of dynamic hybrid lumbar fixators should be designed with less interface stress occurring at the screw-vertebral junction and facet joint to decrease pedicle screw loosening/breakage under various disc degenerations. The high rigidity-raising effect of pedicle static fixators inevitably induces a potential culprit in developing junctional problems attributed to the overconstrained mobility of fixed segments and more load being compensated by the adjacent segments –3. PedicFrom the biomechanical viewpoints, PDS shows a stabilizing effect mainly in flexion/extension and lateral bending but does little to restrict axial rotation on the treated segment . In termx-y-z) was established with the origin at the centroid of L5 constrained bottom to describe the 6 degrees of freedom (df) motion of the lumbosacral column. A total of 45 ligaments and 46 muscles were simulated symmetrically with respect to y-z sagittal plane in this FE analysis [A lumbosacral model had been established and validated by comparison with the cadaveric and numerical data of intact and degenerative models in our previous study . An inteanalysis .To estimate the stiffness of the Isobar damper, a uniaxial material testing system  equipped with a custom-made holder was adapted under a pure compression and distraction loading . The maxReferring to implantation guide with the same screw diameters for all fixations, the Dynesys cord with 300 N pretension was applied through the cannulated spacer, and the Isobar integral damper was executed with a preset 15° lordotic rod of 5.0 mm in diameter at L3-L4 transition segment after static fixation at L4-L5. The Dynesys spacer simulated as a straight tube  assumed to be in intimate contact at the screw spacer interfaces. The spring cord stiffness (origin: 650 N/mm) was modified to investigate the trade-off region of junctional problem , 22. TheThe aforementioned loads and iterative adjustments were applied to control the same value of total disc ROM (by a follower preload) in the models of intact degeneration, 2 static, and 2 hybrid fixations , 23–25. r  was calculated to measure the interpedicular motion  more than at the caudal one (L5-S1) Figures . Both thCompared with a degenerative model without instrumentation at the L3-L4 transition segment, the decreased FCF in the model implanted with Isobar integral damper ranged from 6% (rotation) to 57%(extension), from 45% (bending) to 135% (extension), and from 51% (rotation) to 203% (extension), respectively . The decIn the current L3/L4 mild degenerative condition, the trade-off region of Dynesys cord stiffness was set as 50-200 N/mm because the convergent value of disc stress decussated around 50 N/mm and approached zero around 200 N/mm . In termD1) to 1.2 mm (D3), the constraint (changes in percentage) of ROMs at L3/L4 disc were 11% (from -62% to -51%), 8% (-69% to -61%), 9% (-26% to -17%), and 1% (-8% to -7%), and the constraint of L3/L4 disc stresses were 6% (from -55% to -49%), 4% (-65% to -61%), 5% (-32% to -27%), and 4% (-37% to -33%), in flexion, extension, lateral bending, and axial rotation, respectively. When the axial stiffness of damper decreased from 1200 N/mm (S1) to 400 N/mm (S3), the constraint (changes in percentage) of ROMs at L3/L4 disc were 3% (from -62% to -59%), 3% (-72% to -69%), 8% (-26% to -18%), and 2% (-8% to -6%), in flexion, extension, lateral bending, and axial rotation, respectively, and the constraint of L3/L4 disc stresses displayed little changes (<2%). On average, the MAD induced ROM and disc stress changes (ranged from 9% to 12% in extension and bending) at L2-L3 cephalic adjacent segment more than those at L5-S1 caudal segment. In the current degenerative condition, the MAD effect impact on Isobar performance was more prominent than the axial stiffness effect.The design effect of MAD versus axial stiffness impact on Isobar performance was compared . When thThe screw-bone interface stress distribution at transition level L3 was shown from the bound screw tip to posterior screw hub under the dynamic fixation of original Isobar or DTO device . The peaP > 0.05). On the contrary, the sensitivity of Isobar dynamic performance along the pedicle screw travel path was in close accordance with various disc degenerations. The Isobar revealed significant IPT which were scaled by 0.87 in axial load combined with flexion, and by 0.80 (stretching side) and by 0.50 (contraction side), respectively, in axial load combined with lateral bending (P < 0.05). Concerning the ID behavior, the Isobar revealed a significant difference in vertical axial load combined with flexion and contraction side in axial load combined with lateral bending (P < 0.05) that corresponded to the design effects of damp axial displacement and ±2.25° angular deviation. The ID behavior of DTO revealed significant borderline differences only in pure axial load or combined with lateral bending that corresponded to the design effects of longitudinal cord/spacer without prominent angular deviation.The performance of dynamic motion devices at transition segment was evaluated by calculating the average of IPT and ID in scale . When thThe flexibility of dynamic Dynesys cord spacer or Isobar rod-to-rod damper joint was designed in various extents to promote motion preservation and load-sharing effects on the transition segment. We decreased the stiffness of Dynesys cord or Isobar integral damper within “trade-off region” and found the DTO hybrid fixation afforded ROM and disc stress compensation in flexion more physiologically than the Isobar damper rod. The Dynesys trade-off cord stiffness  increased motion at the transition L3/L4 segment to balance the stresses at the adjacent L2/L3 and L5/S1 discs with convergent values of 17% (from 43% to 26%) for ROM and of 26% (from -3% to 23%) for disc stress, respectively, in flexion . BesidesIn the literature review of FE analysis concerning Dynesys performance, the flexible implant forces depended upon the spring cord stiffness and spacer length (2 mm higher after distraction) to reduce disc stress and posterior annulus bulging –9. The oThe screw spacer linkage acts as a fulcrum leverage with vertebral loads and cord pretension being applied at the two sides. Chien et al.  revealeddf-motions to preserve mobility for load sharing at the treated segment. An assembled dampener contained stacks of wear-resistant discs as shock absorbers to reduce axial stiffness (1/3.6 of static rod) [The Isobar damper joint on a preset lordotic rod allows for 6 tic rod) . Theorettic rod) ) to redutic rod) . The aboThe IPT and ID metrics denoted the relative motion of adjacent pedicle screws at the transition segment. The optoelectric tracking of three-dimensional kinematic behavior has been conducted to find that a longer spacer in Dynesys increased ROM and decreased FCF . We adapDiscrepancies between numeric and experimental results exist owing to the simplification of spinal fixator design in the FE model or difficulty in maintaining longitudinal rod or cord spacer exactly at the same lordotic angle during consecutive experimental tests. Initial preload guaranteed the stability on an instrumented assembly to reduce half the von Mises stress values . The curWith respect to the model limitations, our study assumed the changes in disc-facet strength concurrently without involving lordotic curve progression, facet hypertrophy, endplate sclerosis, osteophytes, and annular tears. Regarding the designs of spinal implant, hyperelastic behavior of cord spacer was not considered, and with only one length of spacer support for FE analysis. Meanwhile, the screw stress may be underestimated because of the lack of a screw thread and simplification of screw configuration. The loosening of bone screw and screw spacer interfaces were focused as major failure modes in dynamic stabilization systems, but the complex interfacial slippage was not easily modeled for the sake of efficiency and convergence of highly nonlinear simulation . This FEDynamic hybrid fixators can protect transition segment but may induce kinematic and mechanical compensation to adjacent segments which was a trade-off problem of the fixed segment. For the Dynesys component, the highly stressed interface of bone screw and screw spacer makes them potentially pullout and fatigue failure. The Isobar damper joint should adjust the maximal allowable displacement with less stress occurring at screw vertebral junction for dynamic fixator use.


Norovirus (NoV) is the most important cause of seafood-borne gastroenteritis worldwide, mainly associated with the consumption of raw oysters. NoV is often present in oysters that comply with existing control standards for shellfish. Therefore, the improvement of post-harvest treatments and practices can represent one of the main strategies to reduce the incidence of viral diseases related to shellfish. This study aimed to investigate long-term relays for the reduction of NoV levels in live oysters, during the high-risk cold months, by transferring the oysters from a more contaminated site to two sites with lower NoV levels. The efficacy of relaying was evaluated by analyzing oyster samples collected at days 0 (T0) and 30 (T30) for NoV levels using a real-time quantitative PCR (RT-qPCR). The NoV level at the relay sites was consistently lower than at the harvest site. The relay process for 30 days in seawater with a lower NoV level resulted in a decrease in the NoV load compared to day 0 with significant reductions depending on the site and genogroup of NoV considered. These results suggest that long-term relaying of oysters to reduce NoV levels is promising and could help growers to improve oyster safety; however, further investigations are needed. The widespread habit of consuming these raw shellfish exposes the consumer to the risk of gastroenteritis. Among the main pathogens responsible for foodborne gastroenteritis are Noroviruses (NoVs) which caused 130 outbreaks in Europe in 2020 [ividuals . Therefoividuals . Wastewaividuals .E. coli. Depuration of shellfish is not effective for reducing enteric viruses, and episodes of viral gastroenteritis associated with the consumption of bivalve mollusks evaluated with the E. coli parameter occur annually around the world [The conventional approach to the purification of oysters envisaged by current legislation is depuration, a process used to remove microorganisms and other contaminants from bivalve mollusks by placing them in tanks of clean seawater, often recycled and disinfected using ultraviolet light, ozone, or other means . Depurathe world . It is the world .Relaying is an alternative treatment to depuration; this involves a longer-term purification process (often requiring ten days or longer) . In relaLimited data on the effectiveness of prolonged relaying for NoV reduction are currently available ,12,13,14Crassostrea gigas) used in this study were harvested from a class B shellfish farming area in northwestern Italy (site 1) and moved to two different sites of category A (site 2 and site 3) (Oysters ( site 3) . Site 1  site 3) . Sixty oAdditionally, samples of 20 oysters were harvested from sites 2 and 3 on the same days as the oyster sampling at site 1 (T0). Further samples of 20 oysters were sampled from sites 2 and 3 on the same days as the oyster sampling after 30 days of relaying (T30). These additional samples were analyzed for NoV GI and NoV GII to monitor the concentration of NoV in native oysters. Seawater parameters, such as temperature and salinity, were recorded at each sampling in site 2 and 3.g. Finally, supernatants were recovered for RNA extraction, and their volumes were recorded.Oyster samples (each composed of ten oysters) were tested according to the ISO 15216-1:2017 method [® platform and the NucliSens magnetic extraction reagents  according to the manufacturer’s instructions. RNA was eluted into 100 μL of elution buffer and was immediately used for the NoV quantification by real-time RT-PCR or stored at −80 °C until real-time RT-PCR analysis.Viral RNA was extracted from 500 µL of the supernatants using the EGENE-UPThe detection and quantification of NoV GI and NoV GII genomes were performed by real-time quantitative reverse transcription PCR (RT-qPCR) according to ISO 15216-1:2017, and the reactions were carried out using the Biorad CFX96TM Real-Time PCR thermocycler . The number of RNA copies per μL of each sample was calculated by matching the sample Cq value to the standard curves (one for each target) created with the tenfold serial dilution of a dsDNA standard for NoV GI and NoV GII. Therefore, the final concentrations were expressed as genomic copies per gram (g.c./g) and were calculated based on the volume of the analyzed extract. According to ISO 15216-1:2017, the Cq value of Mengovirus was obtained in spiked samples and was compared with extracted samples by viral stock to evaluate extraction efficiency. Furthermore, to evaluate the inhibition of RT-qPCR, the Cq value was obtained in samples spiked with 1 µL of external control RNA for both GI and GII and was compared with that obtained in samples without the addition of external control. Results with extraction efficiency > 1% and RT-qPCR inhibition ≤ 75% were considered valid. The LOQ was established by the European Union Reference Laboratory (EURL). The LOQ for NoV GI was calculated as 140 g.c./g and 130 g.c./g for NoV GII.Data were verified for Normality using the Shapiro–Wilk test. The Wilcoxon rank-sum test was used to compare the samples between the two oyster farming areas at time T0. The Wilcoxon matched-pairs signed-rank test was used to compare the equality of matched pairs of observations, i.e., time T0 versus T30.5 viral g.c./g at site 1, 5.5 × 104 viral g.c./g at site 2, and 2.7 × 104 viral g.c./g at site 3. The maximum load of NoV GI was 5.1 × 102 viral g.c./g at site 1, 1.9 × 103 viral g.c./g at site 2, and 6.2 × 103 viral g.c./g at site 3. Moreover, NoV GII was always detected in all sampling, while NoV GI was absent or below the LOQ in some samples at sites 2 and 3. Among the sites used for relaying, site 3 was the one with a lower concentration of both NoV GI and NoV GII compared to site 2 , when it was present at higher concentration at site 2. NoV GII was found at all sites at higher concentrations than NoV GI. The maximum NoV GII load was 3.1 × 10o site 2 a,b.NoV GI levels decreased in all samples below the LOQ of the assay (140 genomic copies per g) at site 2 after 30 days from the transfer of oysters from site 1. Moreover, NoV GI was not detected in two samples after relaying period a. In theBoth NoV GI and NoV GII levels decreased in all oyster samples of site 1 when transferred to site 3 after 30 days of relaying a,b, and p = 0.04) and for Nov GII (p = 0.009). A statistically significant difference was found by comparing the paired data between time T0 and time T30 both for NoV GI (p = 0.001) and for Nov GII (p = 0.01). The comparison between the two consecutive summations also gave a significant result (p = 0.01). The statistical analysis of stratified sampling at relaying area gave the following results: at site 2, only the reduction of NoV GI was significant (p = 0.031), while at site 3, both NoV GII and the sum of NoV GI and NoV GII reduction were significant (p = 0.031).Relays for 30 days in seawater with a lower NoV level resulted in a decrease in the NoV load in oysters compared to T0 at both sites 2 and 3. In particular, logarithmic reductions between 2.34 and −0.2 and between 2.7 and 0.56 were obtained, respectively, for NoV GII and NoV GI at site 2 . AlthougPhysiochemical parameters of seawater during oysters’ relay periods are summarized in In this study, we report the results of prolonged relaying to decrease NoV concentration in oysters as a possible management strategy to reduce consumer exposure to NoV in the winter season. To study NoV reduction, we used environmentally contaminated oysters from approved production areas classified as Class B areas and transferred them to two different sites with less contamination classified as Class A during the high-risk winter period. NoV monitoring at the main production sites (site 1) and the relaying sites (sites 2 and 3) confirmed that oysters at the main production site contained, on average, higher NoV concentrations.In our study, the presence of high levels of NoV at site 1, compared to sites 2 and 3, is probably related to the characteristics of the area where the oysters are raised. The oyster farm located at site 1 is inside a dam, which protects the commercial port from the open sea by reducing sea currents and favoring the stagnation of contaminants in this area. Whereas in oyster farms located at sites 2 and 3, the depth of the seabed and the distance from the coast favor the dilution and quantity of contaminants present in marine waters and reduce the risk of contamination of oysters.The data presented here indicate that oysters from the main collection site significantly reduced their NoV concentration by relaying for 30 days in seawater sites with a lower NoV concentration. In only one case, the NoV GII concentration increased after 30 days at site 2 b. The inSo far, few studies have reported a significant reduction in NoV load from oysters relayed in seawater over an extended period. In one case, oysters from the harvesting area responsible for NoV outbreaks were moved to a seawater site free from sewage contamination and maintained there for 17 days . In anotThe ideal procedure for obtaining oysters safely would be harvesting them in areas that are not subjected to any type of contamination. However, very few such areas really exist, and also the waters classified as class A are contaminated by NoV. Moreover, access to clean relaying waters may be a challenge in the future, with an increasing global population. To overcome these problems, long-term relaying could be a mitigation strategy for the enteric virus reduction; if possible, move the oysters from areas with high levels of NoV to areas with a lower viral concentration in order to reduce the risk to consumers as much as possible. Indeed, in some studies, it is observed that the likelihood of becoming infected with NoV increases with NoV dose ,19,20,21In our study, the obtained dataset provides statistically significant differences between the different sites studied and between the different genogroups and gives an initial indication of the trends and effects of this treatment. However, further studies during winter seasons may be useful to integrate the data presented here. The limitation of our study was the lack of Norovirus-free marine areas to move the oysters to for relaying since this strategy would be the best for studying the oyster relaying. Unfortunately, in our case, no NoV-free marine areas are close to the main production site where the oysters could be transferred at affordable costs before sale. As discussed above, it is increasingly difficult to find NoV-free marine waters, and, as in our case, waters classified in class A are also usually contaminated by NoV, albeit in a lower concentration than the sites classified in class B. Therefore, in our study, we chose to move the oysters from a more NoV-contaminated site to less NoV-contaminated sites in order to study the long-term relay for the reduction of NoV concentrations in oysters.In conclusion, the results of this study suggest that relaying naturally contaminated oysters to sites close to the main production area with lower NoV concentration before their final harvest could provide a practical and low-cost mitigation strategy to reduce NoV in oysters. Relaying could lead to a decrease in the risk of NoV disease from oyster consumption during the colder season when NoV is present in high concentrations. This study may help growers to improve both the marketability and safety of oysters, as well as provide additional information relevant to risk management decisions for regulatory agencies in light of the potential introduction of a statutory limit.


Through tightly controlled multilayer mechanisms, vascular endothelial growth factor receptor-2 (VEGFR-2) activation and its downstream signal transduction govern vasculogenesis and pathological angiogenesis, such as tumor angiogenesis. Therefore, it is critical to understand the molecular mechanisms governing VEGFR-2 signal transduction. We report that protein arginine methyltransferase 4 (PRMT4) via its highly conserved EVH1 and PH domain-like N-terminal domain binds to VEGFR-2 and mediates methylation of the juxtamembrane arginine 817 (R817) on VEGFR-2. Methylation of R817 selectively increases phosphorylation of tyrosine 820 (Y820). Phosphorylation of Y820 facilitates the c-Src binding with VEGFR-2 via Src homology domain 2 (SH2). Interfering with the methylation of R817 or phosphorylation of Y820 inhibits VEGFR-2-induced filopodia protrusions, a process that is critical for the core angiogenic responses of VEGFR-2. Methylation of R817 is an important previously unrecognized mechanism of the angiogenic signaling of VEGFR-2, with implications for the development of novel-targeted VEGFR-2 inhibitors.  •Arginine 817 methylation regulates phosphorylation of Y820 on VEGFR-2•Phosphorylation of Y820 recruits c-Src kinase to VEGFR-2, leading to its activation•VEGFR-2/c-Src axis mediates filopodia protrusions in endothelial cells Biochemistry; Molecular physiology; Cell biology Vascular endothelial growth factor receptor-2 (VEGFR-2) is a key receptor tyrosine kinase (RTK) that plays a major role in vasculogenesis and angiogenesis. It stimulates various cellular responses in endothelial cells, including cell proliferation, survival, migration, and differentiation . VEGFR-2Our previous study identified five methylation sites on VEGFR-2, including Arg817, Lys856, Lys861, Lys1041, and Arg1115 . Lys1041In this study, we have focused on the methylation of Arg817 of VEGFR-2 and demonstrate that PRMT4 catalyzes methylation of VEGFR-2 at Arg817. Methylation of Arg817 modulates phosphorylation of Tyr820, increases the recruitment of Src kinase to VEGFR-2, leading to c-Src activation. Interfering with the methylation of R817 inhibited VEGFR-2-induced filopodia protrusions, a key cellular event that is required for VEGFR-2-induced angiogenic signaling.c-Myc antibody. The result showed that all the PRMTs (1–8) were co-immunoprecipitated with VEGFR-2 with or without VEGF stimulation  and three lysine sites  . Methylamulation B, indicamulation B.Figure in vitro GST-pulldown assay. Other PRMTs could also interact with VEGFR-2, albeit weakly.The observation that all the PRMTs were co-immunoprecipitated with VEGFR-2 suggested to us that PRMTs either share a common motif/domain that enables their interaction with VEGFR-2 or the observed interaction of PRMTs with VEGFR-2 occurs in a non-specific manner. To address the specificity of the binding of PRMTs with VEGFR-2, we generated a panel of GST-fusion recombinant PRMTs C and tesTo identify the potential region on PRMTs involved in the interaction with VEGFR-2, we aligned the amino acid sequence of PRMTs. There was a significant degree of homology in the N-terminus of PRMTs A. HoweveTo examine the hypothesis that the N-terminal of PRMT4 mediates the binding of PRMT4 with VEGFR-2, we generated two truncated PRMT4 constructs encompassing the N-terminal alone (PRMT4-1-142) or PRMT4 without the N-terminus (PRMT4-143-585) C. We co-in vivo methylation assay via 3H-S-adenosyl methionine (3H-SAM) labeling. The result showed that in the context of overexpression PRMT4, VEGFR-2 is methylated in vivo, and mutation of R817 significantly inhibited PRMT4-dependent methylation of VEGFR-2 . Western blot analysis of the whole cell lysates of VEGFR-2/HEK-293 and A817/VEGFR-2/HEK-293 showed that phosphorylation levels of Y820 on the A817/VEGFR-2 were significantly reduced compared to the phosphorylation of Y820 on the wild-type VEGFR-2 F. Next, in vitro Src kinase assay  in an se assay D. Moreovse assay D. Phosphse assay D, indicase assay E, we exase assay F. Consis reduced G. Taken The key angiogenic responses of VEGFR-2 activation such as capillary tube formation, sprouting, and cell migration involve filopodia protrusions of endothelial cells. Similarly, activation of Src family kinases plays a central role in these finely regulated cellular events. Considering the effect of methylation of R817 on the phosphorylation of Y820 and Src activation, we investigated the ability of mutant R817 and Y820 VEGFR-2 constructs expressed in HEK-293 cells in filopodia protrusions. Our analysis showed that stimulation of HEK-293 cells expressing VEGFR-2 with VEGF induced robust filopodia-like structures as detected by phalloidin staining of the cells . HoweverVEGFR-2 is predominantly expressed in endothelial cells; therefore, we decided to validate our findings in endothelial cells. Our results showed that treatment of HUVEC-TERT cells with SU6656, a potent Src kinase inhibitor, selectively inhibited phosphorylation of Y820 on VEGFR-2 but not Y1054 A, indicaIn this study, we provide evidence that R817, a proximal residue to Y820 on VEGFR-2, is methylated and this methylation modulates phosphorylation of Y820. PRMT4 through its N-terminal domain interacts with VEGFR-2 and mediates methylation of R817. Methylation of R817 enhances phosphorylation of Y820 and facilitates the recruitment of c-Src to VEGFR-2, leading to increased Src kinase activation and VEGFR-2-induced filopodia formation. Our previous study demonstrated that methylation of a conserved lysine 1041 (K1041) on the VEGFR-2 plays an essential role in the kinase activation of VEGFR-2 by regulating the key autophosphorylation tyrosine 1052 (Y1052). Demonstration that methylation of R817 selectively modulates Y820 phosphorylation and Src recruitment to VEGFR-2 indicates significant crosstalk between the arginine and lysine methylation and tyrosine phosphorylation of VEGFR-2. Interestingly, all the PRMTs (eight PRMTs) tested for their ability to interact with VEGFR-2 in HEK-293 cells, showed a strong binding. However, under more stringent conditions, only PRMT4 strongly interacted with VEGFR-2. One of the distinct features of PRMT4 is the presence of EVH1 and PH domain-like N-terminal domain. We demonstrate that this unique N-terminal domain of PRMT4 specifically interacts with and mediates the binding of PRMT4 with VEGFR-2.Another important aspect of our study is the demonstration of phosphorylation of Y820 and its role in the recruitment of c-Src kinase to VEGFR-2. Although previous studies have shown that VEGFR-2 mediates c-Src activation , howeverEmerging evidence indicates that PRMTs play broad functional roles in the regulation of endothelial cell function and angiogenesis. For example, endothelial-specific loss of PRMT1 in mice resulted in angiodysplasia , and PRMOur study presented here demonstrates that R817 methylation modulates phosphorylation of Y820, which leads to the recruitment of c-Src kinase and its activation by VEGFR-2. VEGFR-2/Src pathway derives the formation of filopodia protrusions in endothelial cells, which is important for the coordinated angiogenic processes such as sprouting and cellular migration. However, considering that multiple PRMTs can interact with VEGFR-2, it is likely that other PRMTs can mediate the methylation of R817. The role of other PRMTs in the methylation of R817 other than PRMT4 is not analyzed in this study. Moreover, the potential mechanisms by which methylation of R817 modulates phosphorylation of Y820 require further investigation. Methylation of R817 could regulate phosphorylation of Y820 either by altering the activity of a particular tyrosine phosphatase (PTPase) or a tyrosine kinase toward dephosphorylation or phosphorylation of Y820, respectively, but we have not examined these possibilities. Moreover, the direct role of R817 methylation in the VEGFR-2-mediated angiogenesis such as tumor angiogenesis requires further investigation.nrahimi@bu.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Nader Rahimi  were purchased from ATCC (Cat# CRL-1573), porcine aortic endothelial cells (PAE) and TERT transformed human umbilical vein endothelial cells (HUVEC-TERT) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (50 units/mL) and streptomycin (50 mg/mL). PAE and HUVEC-TERT cells are previously described . Other k820DASKWEF) was used to develop polyclonal rabbit anti-phospho-Y820 antibody. The pY820 antibody was affinity purified via Protein-A Sepharose. The specificity of the antibody was further validated using HEK-293 cells expressing VEGFR-2 or mutant Y820-VEGFR-2. The anti-pY820 antibody did not react with the mutant Y820-VEGFR-2, indicating that it specifically recognizes phosphorylated Y820.Purified phospho-peptide corresponding to tyrosine 820 to human VEGFR-2 (DELPLDEHCERLPpYHEK-293 cells stably expressing VEGFR-2 were transfected with PRMT constructs via PEI (polyethylenimine) as described . After 42. Viral production was achieved by transfection into 293-GPG cells, and viral supernatants were collected for 5 days; viral medium was then used as described , and normalized whole-cell lysates were subjected to immunoprecipitation by incubation with appropriate antibodies as shown in the figure legends. Immuno-complexes were captured by incubation with either protein A-Sepharose or protein G Agarose beads. The samples were boiled for 5 min at 95°C, the immunoprecipitated proteins were subjected to western blot analysis. In some cases, membranes were stripped by incubation in a stripping buffer  at 50°C for 30 min, washed in Western Rinse buffer (20 mM Tris and 150 mM NaCl), and re-probed with the antibody of interest. The blots were scanned and subsequently quantified using ImageJ (NIH).32P]. After 30 min of incubation at 30°C, reactions were terminated by the addition of 2× SDS sample buffer, and samples were subjected to SDS-PAGE. The SDS-PAGE gel was fixed in 45% methanol and 10% acetic acid for 30 min at room temperature and dried at 80°C under a vacuum and autoradiographed.The Src kinase assay was performed as described previously. Briefly, lysates from HEK-293 cells expressing wild type VEGFR-2 or mutant VEGFR-2 constructs were prepared, and equal protein amounts from each cell lysate were immunoprecipitated with anti-Src antibody (Santa Cruz Biotechnology) and half of each immunoprecipitate was assayed for Src kinase activity by incubating with 10 μL of reaction buffer , 2 μg of freshly prepared acid-denatured enolase (Sigma) min, and 10 μCi of [γ-In vitro methylation assay was performed as described with minor modification (fication . BrieflyEscherichia coli transformed with PRMT-pGEX-4T2 constructs. Single colony was grown in 5 mL Luria-Bertani (LB) medium overnight at 37°C. The culture was then expanded into 300 mL LB medium until an optical density of 0.4–0.6. The protein expression was induced by 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) at 30°C for 4–15h. The cells were collected and re-suspended in GST buffer , 150 mM NaCl) and sonicated (4 cycles/5 s each). After centrifugation, the supernatant was incubated with glutathione Sepharose beads for 2 h at 4°C and subsequently washed four times before use.Recombinant PRMT proteins were purified from the BL21(DE3) In vitro GST fusion PRMTs or Src-SH2 binding experiments were performed as described previously (eviously . BrieflyHEK-293 cells or PAE cells expressing VEGFR-2 or mutant VEGFR-2 constructs were seeded onto coverslips and grown overnight in 60-mm plates to 70–90% confluence. Cells were serum starved overnight and then either left unstimulated or stimulated with VEGF (100 ng/mL) for 30 min. Cells were washed once with PBS and immediately fixed with freshly prepared 4% paraformaldehyde for 15 min at room temperature. After being washed three more times with PBS, the cells were permeabilized with 0.25% Triton X-100 in Western rinse buffer for 10 min at room temperature and then washed three times with PBS. Cells were stained with Phalloidin-FITC and DAPI and the slides were viewed under the Nikon Deconvolution microscope equipped with a camera and 3-4 pictures were taken per group. Quantification of filopodia was carried out using open-source software, filopodyan as previously described .Experimental data were subjected to a Student’s t test or one-way ANOVA, where appropriate, with a representation of at least three independent expderiments. p < 0.05 was considered significant, or as indicated in the figure legends.


The prevalence of non-immunoglobulin E (IgE) mediated food allergy is poorly established outside of cow’s milk allergy, with a challenge-proven adjusted incidence ranging between 0.13 and 0.72%. The presence and presentation of non-IgE mediated allergy in exclusively breastfed infants is highly debated. The dilemma this poses for healthcare professionals and parents, is on the one hand the unwarranted elimination and therefore health risk to the breastfeeding mother and on the other hand under-recognition of a food allergen being a culprit in the non-IgE mediated symptoms of breastfed infants. Current international guidelines recommend exclusive breastfeeding ideally until ~ 6 months of age and the German guidelines 4 – 6 months. It is also acknowledged that breastfeeding should be promoted also within the population of food-allergic infants. This review paper aims to assess non-IgE mediated food allergies in breastfed infants using an evidence-based approach and provides clinicians working with these patients with practical guidance.  The prevalence of non-immunoglobulin E (IgE) mediated food allergy is poorly established outside of cow’s milk allergy (CMA). The EuroPrevall Study, based on over 12,000 infants from different European countries, found a challenge-proven adjusted incidence ranging between 0.13 and 0.72% [There is limited data of non-IgE mediated allergy involving other common allergens in children . Although this is reported in observational studies and healthcare professionals working in food allergy also have made this observation, the elimination of these food allergens is often based on experience rather than clinical research . The presence of food allergy in exclusively breastfed infants remains a highly debated topic. The most commonly quoted study in breastfed infants is on CMA by Høst et al.  from 198At the heart of the debate of non-IgE mediated allergy in breastfed infants, is the question to whether maternally consumed food allergens can lead to symptoms. A recent scoping review found 27 studies assessing bovine milk protein in breastmilk that included data on the type of sampling method, the sampling time, the lactation stage, maternal allergy status, and, most importantly, the impact on the infant . This puThe correlation of clinical manifestations and β-lactoglobulin does however not prove causality. This was argued by Munblit et al.  in 2020,There are several problems with the aforementioned theoretical calculations. The first and most obvious is that the ED01 and ED05 have been based on IgE mediated allergy. Additionally, the testing method, maternal atopy, and the pathophysiology of non-IgE mediated allergy has not been considered in calculating potential reactions of cow’s milk protein through breastmilk. Studies have shown marked difference in human breastmilk between allergic versus non-allergic mothers, including the levels of short-chain fatty acids , which iAll of the aforementioned studies discuss the presence of bovine β-lactoglobulin in breastmilk and the potential to cause non-IgE mediated CMA, but other allergens such as soya, wheat, and egg have been detected in breastmilk and therefore also have the potential to elicit non-IgE mediated food-allergic reactions, but data to substantiate this is sparce and primarily based on clinical experience , 23. The diagnosis of non-IgE mediated allergy in breastfed infants is further complicated, because many of the symptoms  overlap with pediatric functional gastrointestinal disorders (FGID) and other commonly occurring childhood diseases. Vandenplas et al.  performeThere are currently no recommended biomarkers for the diagnosis across the spectrum of non-IgE mediated allergies, which includes the breastfeeding infant presenting with these delayed symptoms. Some studies have proposed atopy patch testing to determine “delayed sensitisation” , howeverIgG and IgG4 testing have also been suggested to support a diagnosis of non-IgE mediated allergy, but these tests have little established clinical validity and their use is also not recommended in routine clinical practice , alongsiWhilst the absence of specific IgE is an expected characteristic of non-IgE mediated food allergies, some children may present with overlapping disease and allergic co-morbidities associated with IgE sensitization  , 40. ThiFinally, endoscopy is commonly reported in research related to the diagnoses of various non-IgE mediated allergic conditions, including EoE, where this is an essential diagnostic tool. As this procedure can be technically difficult in young infants and requires full anesthesia, the EAACI position paper suggests that endoscopy only be performed when there is a strong suspicion of an alternative diagnosis or unremitting symptoms . The maternal dietary elimination of offending allergens remains the mainstay for dietary management of non-IgE mediated allergies in breastfed infants. The most commonly reported allergen is cow’s milk, but a non-IgE mediated allergy in breastfed infants to soya, egg, and wheat has also been described , 43. AllThe length of a diagnostic elimination diet in non-IgE mediated food allergies varies according to guidelines but is usually between 2 and 4 weeks. Clinicians need to also take into account that circulating β-lactoglobulin can be detected up to 7 – 10 days after the elimination . LozinskThe updated iMAP guidelines on non-IgE mediated allergies, recommend in exclusively breastfed children that cow’s milk products should be reintroduced in the mother’s diet in previously consumed amounts over a 1-week period . The latFor breastfed infants with non-IgE mediated allergies who are already on solids, there is also very limited data. The iMAP guidelines provide a consensus-based milk ladder, which has been constructed on the existing data that heating and fermentation reduces the allergenicity of cow’s milk , 51, 52.Non-IgE mediated allergies in breastfed infants remains a complex diagnosis that needs careful clinical consideration. There is the concern of both over- and underdiagnosis and most importantly the nutritional risk for the mother when unwarranted elimination diets are used. None. Academic lectures for Danone/Nutricia, Mead Johnson, Nestle, and Abbott. Consultancy for Else Nutrition and on the CoMISS board for Nestle Health Science.


The freezing of water into ice is a key process that is still not fully understood. It generally requires an impurity of some description to initiate the heterogeneous nucleation of the ice crystals. The molecular structure, as well as the extent of structural order within the impurity in question, both play an essential role in determining its effectiveness. However, disentangling these two contributions is a challenge for both experiments and simulations. In this work, we have systematically investigated the ice-nucleating ability of the very same compound, cholesterol, from the crystalline (and thus ordered) form to disordered self-assembled monolayers. Leveraging a combination of experiments and simulations, we identify a “sweet spot” in terms of the surface coverage of the monolayers, whereby cholesterol maximises its ability to nucleate ice  by enhancing the structural order of the interfacial water molecules. These findings have practical implications for the rational design of synthetic ice-nucleating agents. The freezing of water into ice is still not fully understood. Here, we investigate the role of structural disorder within the biologically relevant impurities that facilitate this fundamental phase transition. Cholesterol is an interesting model system to study since it is an effective ice-nucleating agent and a number of steroids with molecular structure similar to CHL show equally strong ice-nucleating ability in their crystalline phases.38,39 In this work, we have isolated the impact of structural order only, by systematically investigating the ice-nucleating ability of the very same compound, cholesterol, from the crystalline (and thus ordered) form to disordered self-assembled monolayers, thus focusing in detail on the impact of structural order on heterogeneous ice nucleation without having to compare the ice-nucleating efficiency of different materials.Here, we answer this crucial question by adopting a different approach, namely probing the ice-nucleating ability of the same substance, cholesterol (CHL), in different forms, from its crystalline phaseBy bringing together state-of-the-art experimental techniques and atomistic computer simulations, we find that CHL monolayers display a weaker ice-nucleating activity compared to CHL crystals. A systematic investigation of CHL monolayers characterised by different coverages  reveals that the degree of order of the CHL molecules within the monolayers has a direct impact on the population of pre-critical ice nuclei forming at the CHL–water interfaces. In particular, there exists a specific interval of CHL coverages that maximises the ability of this steroid to order interfacial water molecules in such a way as to facilitate ice nucleation.This systematic investigation of the impact of order and disorder on the ice-nucleating ability of a biologically relevant compound allows us to unravel the microscopic motivations underpinning its activity and offer valuable practical guidelines toward the rational design of the next generation of INAs. In particular, our results imply that more emphasis should be placed on the supra-molecular aspects of biological INAs as opposed to their molecular structure.40 Droplets of pure water (30 μL volume) were pipetted into sterile 384-well plates  by a liquid handling station . Pure water was prepared as described in ref. 2 mol−1 to 4 Å2 mol−1, respectively, assuming that all cholesterol remains at the water surface and does not diffuse into the bulk. Of each concentration, 96 droplets were measured, and experiments were performed from 0 °C to −30 °C at a cooling rate of 1 °C min−1. The temperature uncertainty was ±0.2 °C.40 As controls, 30 μL droplets of pure water, and 30 μL droplets of pure water with 1 μL droplets of pure chloroform were measured. From the fraction of total droplets frozen, we have computed the cumulative number of nucleation sites per cholesterol molecule, referred to as nmol, which allows us to compare the ice-nucleating ability of the different CHL monolayers with that of CHL crystals. The details of the nmol analysis can be found in the ESI.nmol, the stronger the ice nucleation activity of the sample (per molecule). Confidence intervals were calculated using Monte Carlo simulations according to ref. nmol values. The confidence intervals are the 10th to 90th percentile range of the simulated values. Due to the relatively high number of droplets used in these experiments the confidence interval is relatively narrow.Freezing experiments of the CHL monolayer were performed using the high-throughput twin-plate ice nucleation assay (TINA).via the CHARMM36 force field42,43 and the TIP4P/Ice44 model, respectively. This specific combination has been validated on multiple occasions within the recent literature: not only has it been demonstrated to provide an accurate description of supercooled water and ice at the interface with biological material,45–47 but we have also explicitly verified its reliability for water and ice in contact with cholesterol.33Cholesterol and water molecules were both modelled at the atomistic level 42,48 has been used to perform molecular dynamics (MD) simulations within a variety of ensembles, including the NγT ensemble (with constant surface tension γ)49 so as to take into account different CHL coverages . A leap-frog algorithm50 has been used to integrate Newton's equations of motion with a time step of 2 fs. A twin cutoff of 12 Å has been used for both electrostatic and van der Waals interactions, where, for the latter, forces have been smoothly switched to zero between 10 and 12 Å. The Bussi–Donadio–Parrinello thermostat51 has been used to sample the canonical ensemble, in conjunction with the Berendsen barostat (in a semi-isotropic fashion given the slab geometry)52 if performing simulations in the NPT or NγT ensemble. The coupling constants for the thermostat and barostat are 0.5/1.0 (CHL/water) and 4.0 ps, respectively. The LINCS53 and SETTLE54 algorithms have been used to constrain the CHL bonds involving hydrogen atoms and to enforce the geometry of water molecules, respectively.The GROMACS package (version 5.1.4)z-axis  in-plane dimensions of the simulation box along the compression protocol ranged from 67 (SA/mol ∼ 70 Å2) to 44 Å (SA/mol ∼ 30 Å2). The z-dimension of the simulation box was set to 200 Å, roughly three times the extent of the CHL/water system along the z direction, thus allowing for a substantial vacuum region as suggested by the protocol detailed in ref. SA/mol ∼ 70 Å2) of roughly 40 Å. We note that even at the lowest value of SA/mol we have simulated, the in-plane dimensions of the simulation box are significantly larger than (twice) the extent of the pre-critical ice nuclei we have observed,56 which can exceptionally span up to 16 Å in any given direction.The , while γwater–CHL–vacuum can be written as:Lz and Pαα correspond to the length of the simulation box along the z-axis and the diagonal component of the pressure tensor along the α coordinate.A quantity of special interest when dealing with self-assembled monolayers at aqueous interfaces is the surface pressure, which in this case can be obtained as:SA/mol in order to eliminate the effect of pressure fluctuations: after 50 ns of equilibration, each configuration has been quenched from 300 to 230 K in 50 ns following a linear quenching protocol. These configurations have finally been used as the starting point for the long (≈1.4 μs) production runs used to obtain the results reported in this work to study the formation of ice nuclei. We note that we validated this quenching protocol in ref. S = TM − T = 40 K) to investigate icy water for the TIP4P/Ice water model (the melting temperature Tm of TIP4P/Ice is 270 ± 3 K).62NVT simulations were performed at selected values of et al.,63 which in turn leverages the so-called Steinhardt order parameters.64 We have recently reviewed the full procedure, based on the open-source PLUMED software65 in ref. 67Molecules belonging to ice nuclei have been identified thanks to a clustering algorithm based on the local order parameter introduced by Wang −1 using a Fabry–Perot etalon. The resulting 800 nm beam had a pulse energy of 20 μJ. All sample spectra were collected in SSP (SFG-visible-IR) polarization with incident angles of 34° and 36° for the visible and IR beam, respectively. To reduce heating effects from the laser, the trough containing the CHL monolayer on water was continuously rotated  generating 800 nm pulses with a repetition rate of 1 kHz and a duration of ∼40 fs. Part of the output was used to generate broadband infrared pulses in an optical parametric amplifier  with subsequent difference frequency generation. The IR pulse energy at the sample was approximately 3 μJ. The other part of the laser output was spectrally narrowed to 15 cmsee ref. . To prevvia Brewster angle microscopy as well  was dissolved in chloroform to a concentration of 0.64 mM; all solutions were prepared in a glove box to avoid sample oxidation. To form a monolayer on water, a well-defined number of drops were deposited onto the water surface to get a specific surface coverage , using a microliter syringe (from Hamilton) with a droplet size of 0.5 μL. The surface pressure was monitored throughout the experiment using a Kibron surface tensiometer. The CHL layer was monitored nmol, see the Methods section) for CHL crystals33 and for self-assembled CHL monolayers characterised by different values of SA/mol. It is evident that the same compound shows a much stronger ice-nucleating ability in its crystalline phase, while in the form of a self-assembled monolayer, its potency is greatly reduced.Droplet freezing assays provide a quantitative indication of the ice-nucleating ability of different substances. We have employed a site-specific, or singular interpretation of our ice nucleation data to facilitate background subtraction and comparison between measurements on crystalline cholesterol and the cholesterol monolayers. Details of this analysis are provided in the ESI.69 and inorganic ice-nucleating agents such as the mineral feldspar70 often span a similar range of temperature compared to crystalline CHL, with a sharp increase at the onset of nucleation followed by a plateauing at stronger supercooling. This two-step trend is likely caused by different nucleation sites on the surface of CHL crystals, specifically on the (001) hydroxylated surface and potentially active at different degrees of supercooling (as discussed in detail in ref. SA/mol (as discussed in the next section) the CHL–water interface can be considered as largely homogeneous – i.e., there are no defects to be found, as opposed to most crystalline surfaces. Identifying the exact morphology of ice-nucleating sites on crystalline substrates is beyond the capabilities of existing instrumentation  the ice-nucleating ability of CHL monolayers is basically negligible: this regime corresponds to a situation where CHL monolayers “collapse”74 , we have brought together MD simulations and SFG experiments. As discussed in the next sections, such a sweet spot does exist, and it originates from the changes in the microscopic structure of the monolayers at different coverages.Thus, in order to probe whether there exists a “sweet spot” in terms of the ice-nucleating activity of CHL monolayers, within the range of SA/mol, we focus on the degree of order within the CHL monolayers as a function of surface pressure. To this end, we employ both MD simulations and sum-frequency generation (SFG) spectroscopy.Having established that the ice-nucleating ability of CHL monolayers is not only inferior to that of CHL crystals, but also depends on SA/mol. The computational setup is depicted in SA/mol regime, leads to a substantial overestimation of the absolute values of the surface pressure, the qualitative features of the isotherm are in excellent agreement with the experimental data  has been reported in several previous experimental works36,37,77,78 and is due to the monolayer becoming increasingly compact until it collapses. In particular, Rapaport et al.74 have observed, at 36 Å2 mol−1, the formation of a film composed of a trilayer, with a rough upper surface, a smooth, highly crystalline bilayer in the middle, and a more disordered monolayer in contact with the water phase. However, the finite number of CHL molecules in MD simulations only form a disordered monolayer .We start by investigating the surface pressure of the CHL monolayers–water system as a function of SA/mol reported in via the droplet freezing assay discussed in the previous section. We also note that for high values of SA/mol , both experiments and simulations identify negative values of surface pressure. This could be interpreted, in principle, as an indication of metastability  that has been previously reported in the case of low coverages of lipids, polymers and proteins79–81 – but never in the case of CHL.36,37,77,78 However, as in some conditions, we have observed the same effect experimentally even in the absence of CHL  or characterised by relatively ordered domains that fail to cover the entire CHL–water interface. Our simulations are consistent with these features, as illustrated in Fig. S8 of the ESI,SA/mol in greater detail.In order to identify potential structural changes within the CHL monolayers, it is useful to define a molecular axis for CHL molecules. As illustrated in We note that, despite the fact that the surface density of the CHL monolayers at the “sweet spot” and that of CHL crystals is very similar, CHL monolayers are still not quite as ordered as a crystalline surface. As reported in Fig. S8 in the ESI,−1 and around 2950 cm−1. In line with ref. 3 groups in the tail at the C25 side of the molecule  of the z-coordinate of the centre of mass of the largest ice nucleus found in each configuration generated employing long (0.5 μs) MD simulations. The positions of the peaks corresponding to the increase in the nuclei population at the CHL–water interface have been aligned to zero. The distributions have also been normalised so that the baseline corresponding to the population of nuclei within the bulk of the water slab is equal to one. It is evident that, in the case of CHL crystals, there is a very strong tendency for the ice nuclei to form at the CHL–water interface (light blue shaded region in P(COMz − Max) in the interfacial region is about ten times higher than everywhere else. The situation is different for CHL monolayers, where this tendency for the nuclei to favour the CHL–water interfacial region strongly depends on the SA/mol. For very low values of SA/mol (30 Å2 in SA/mol = 40 Å2 in SA/mol (60 Å2 in P(COMz − Max) between the interfacial region and the bulk of the water slabs decreases once more.We have seen in the previous section that the degree of order within CHL monolayers changes dramatically according to their surface area per molecule. Crucially, our MD simulations reveal that these structural changes in the CHL monolayers translate into different propensities for  ice nuclei to form at the CHL–water interface, as illustrated in 30 Å2 in  correspo60 Å2 in  correspo85 is predictive of the ice-nucleating ability of the different systems under consideration. In fact, according to our experimental data  and collapsed (low SA/mol) regions, thus highlighting the correlation between the degree of order within the monolayers and their ice-nucleating activity.These results indicate that in this case, the analysis of the population of pre-critical nucleidata see , CHL cry86–88). As depicted in e.g., ref. e.g., some ice-nucleating proteins where a layer of “structured” water acts as a template for the ice nuclei to grow upon.16,90 In fact, the presence of the –OH amphiphilic functional groups seems to facilitate ice nucleation in a similar fashion to what we have previously reported for CHL crystals,33 where we have also observed the emergence of both cubic and hexagonal ice. In the case of CHL crystals, the symmetry of the –OH network within the amphiphilic heads of the cholesterol molecules is such that the ice nuclei can form a rather ordered ice–CHL interface, where on average one in two water molecules forms a hydrogen bond with the –OH groups of cholesterol.33 However, for CHL monolayers, even at the sweet spot the limited extent of symmetry as well as the inherent surface roughness of the system lead to the rather disordered ice–CHL interface depicted in Interestingly, when we examine the nature of the pre-critical nuclei at the level of supercooling considered in our MD simulations, there is a coexistence of cubic and hexagonal ice nuclei  in z-axis is measured. The integrated SFG intensity can thus directly be compared to the θz values of the MD simulation. Interestingly, our MD simulations allow us to disentangle the contributions of –OH fragments belonging to either water or CHL molecules. While θz(μCHLOH) displays only a marginal variation as a function of SA/mol, θz(μ2OH) shows a sharp increase in the CHL-ordered region. This trend is in excellent agreement with the experimental data and demonstrates that the increase in the SFG signal is indeed due to the ordering of interfacial water as the CHL monolayers become more ordered.The SA/mol = 70 and 30 Å2, respectively), with the result obtained for a value of SA/mol (40 Å2) corresponding to the highly ordered region of CHL illustrated in θz(μ2OH) ∼ 102°) where one of the hydrogen atoms points toward the CHL surface, leaving the oxygen atom available to create a hydrogen bond with an –OH group of the CHL. A similar configuration produces a shoulder in the probability density at θz(μ2OH) ∼ 130°, which remains unchanged with the value of SA/mol. However, the population of the feature at θz(μ2OH) ∼ 55° substantially decreases for CHL coverages corresponding to the highly ordered region highlighted in θz(μ2OH) ∼ 102° configuration increases. In the proximity of the sweet spot in terms of CHL SA/mol, interfacial water molecules are on average more likely to be able to fully leverage the amphiphilic character of the –OH group of CHL molecules. This is because the ∼102° orientation allows for either or both (a) a hydrogen bond between water and CHL where the water act as a donor, and; (b) a hydrogen bond between water and CHL where the water act as an acceptor. On the contrary, the ∼55° orientation only allows for a hydrogen bond between water and CHL where the water act as a donor. Thus, at the sweet spot, water molecules are on average more likely to interact more effectively with the CHL monolayer, which in turn facilitates the emergence of the OH (from CHL)–water rings network responsible for the formation of both double-diamond and hexagonal ice cages – in a similar fashion to what we have observed for crystalline CHL.33 This subtle orientational change leads to a higher degree of order, as illustrated in Our simulations also offer the opportunity to investigate the changes in the orientation of interfacial water as a function of CHL coverage. In particular, we have chosen to compare the results obtained at low and high coverage  CHL monolayers order interfacial water to an extent sufficient to trigger ice formation. This sweet spot, in terms of ice-nucleating activity, cannot be detected via the frozen droplet measurements reported in SA/mol. However, both our MD and SFG results successfully identify this subtle trend, which is due to the fact that CHL molecules are clearly best packed and most ordered within the crystalline phase. The SA/mol of CHL monolayers can influence their ice-nucleating ability to a great extent, but their degree of order  is still lower than that achieved by crystalline packing. We note that simulating a perfectly ordered monolayer, where the CHL molecules are constrained to their equilibrium positions within the crystalline structure of CHL, corresponds to the computational setup we have used in ref. e.g. phloroglucinol dihydrate monolayers92 as well as wurtzite-structured surfaces.93Thus, it appears that in a specific interval of 30–34 However, it is not clear whether molecular structure outweighs order – or the other way around, chiefly because disentangling the two is a very challenging task for both experiments and simulations.The freezing of water into ice is, at its very core, a question of which particular impurity can facilitate heterogeneous ice nucleation and how. Recent evidence suggests that amongst the different microscopic factors playing a role in this process, the interplay between the molecular structure and the degree of order of an impurity might be the key.In this work, we have been able to tackle this open question by monitoring and rationalising the ice-nucleating ability of the same compound, cholesterol (CHL), from crystals to self-assembled monolayers with varying degrees of order.We have found that CHL crystals outperform any self-assembled CHL monolayer we have investigated in terms of ice-nucleating ability. In addition, our results demonstrate that the more ordered these monolayers are, the better they can facilitate the formation of ice. In fact, we argue that an analysis of the pre-critical ice nuclei, as obtained by means of unbiased molecular dynamics simulations, can be used, in conjunction with experimental measurements such as SFG, to infer the actual ice-nucleating ability of a given material.Crucially, conventional droplet freezing assays often struggle to identify useful trends when dealing with compounds with relatively weak ice-nucleating activity, such as CHL monolayers. One could argue that weakly active INA are of limited practical interest. However, identifying and understanding the changes in their ice-nucleating ability as a function of their structural properties is essential to uncover novel guidelines for the rational design of synthetic INAs.In particular, by bringing together state of the art experimental and computational techniques, we have traced the origin of this ice-nucleating sweet spot to the molecular-level details of the CHL–water interface: less dense CHL monolayers are more disordered  and less effective in modifying the ordering of interfacial water, which in turn enhances the ice-nucleating ability of the CHL substrate.94 and there are many examples of ice-nucleating impurities in the form of self-assembled monolayers of alcohols.95–97 In addition, the formation of ice in biological matter, which is key in the context of cryopreservation, does involve the formation of ice on/through the lipid bilayers which form the building blocks of cells.98–101 Control of ice nucleation very often improves the outcomes of cryopreservation procedures76,102 and, critically, the origin of ice formation inside cells remains very poorly understood.103 Improved understanding of the mechanism of heterogeneous ice nucleation by biological substances has the potential to both facilitate synthesis of new and effective biocompatible ice nucleators and shed light on the start of damaging intracellular ice formation.While this work focuses entirely on CHL, we expect our findings to be directly applicable to similar systems: for instance, particles containing long-chain fatty acids are of great relevance to the formation of ice driven by sea spray aerosols,1,15,104–106 In fact, some of them harness already supra-molecular structures with variable degrees of ordering (ice-nucleating proteins being an obvious example107–111) to facilitate ice formation. At the moment, there is a strong emphasis on the molecular structure of these compounds as opposed to their topology: our results suggest that we might want to shift our attention toward the design of supramolecular assemblies as opposed to trying to discover the most effective functional groups that are supposed to trigger ice formation – often a trial-and-error process.Importantly, the observation that order plays an important role in heterogeneous ice nucleation, has a strong impact on the design and discovery of the next generation of ice-nucleating agents, many of which are bound to be biological in nature.e.g., systematic studies of the ice-nucleating ability of supramolecular assemblies with a strong potential to be leveraged as effective ice-nucleating agents.Indeed, while playing with molecular structure is relatively straightforward , acting on supra-molecular topology and particularly the degree of order within it is a much more complex task, with similar compounds displaying entirely different solid-phase morphologies, not to mention the incredibly challenging characterisation of the ice-nucleating sites in the case of crystalline ice-nucleating agents. While the solution of the puzzle offered by heterogeneous ice nucleation might not be within our reach yet, this investigation provides an essential piece that we hope will foster future work aimed at, https://wrap.warwick.ac.uk/164520/.Data for this paper are available at: G. C. S. performed the molecular dynamics simulations. P. S. and E. H. G. B. performed the sum frequency generation spectroscopy measurements. A. T. B. performed the droplet freezing assays. All the authors contributed to the analysis and the interpretation of the results as well as to the writing of the manuscript.There are no conflicts of interest to declare.SC-013-D1SC06338C-s001