Herbivores large and small need to mechanically process plant tissue. Their ability to do so is determined by two forces: the maximum force they can generate, and the minimum force required to fracture the plant tissue. The ratio of these forces determines the relative mechanical effort; how this ratio varies with animal size is challenging to predict. We measured the forces required to cut thin polymer sheets with mandibles from leaf-cutter ant workers which vary by more than one order of magnitude in body mass. Cutting forces were independent of mandible size, but differed by a factor of two between pristine and worn mandibles. Mandibular wear is thus likely a more important determinant of cutting force than mandible size. We rationalize this finding with a biomechanical analysis, which suggests that pristine mandibles are ideally ‘sharp’—cutting forces are close to a theoretical minimum, which is independent of tool size and shape, and instead solely depends on the geometric and mechanical properties of the cut tissue. The increase of cutting force due to mandibular wear may be particularly problematic for small ants, which generate lower absolute bite forces, and thus require a larger fraction of their maximum bite force to cut the same plant.This article is part of the theme issue ‘Food processing and nutritional assimilation in animals’. DespiteAtta vollenweideri leaf-cutter ants.Based on a simple scaling argument, the maximum available force is expected to increase in proportion to a characteristic area, or with body mass to the power of two-thirds . HoweverAtta species [Leaf-cutter ant colonies consist of up to several million workers, which cover a large range of body sizes, from less than a milligram to over 100 mg in some  species –21. Nota species –25. To c species ,27, see  species ,28). Int species ,28–30, a species  and fora species ,33,36–38A. vollenweideri leaf-cutter ants [Fb ∝ m0.90, in substantial excess of the isometric prediction, Fb ∝ m0.67: a large forager of 40 mg generates peak bite forces of about 800 mN, 16 times more than a small forager of 2 mg, Fb ≈ 50 mN, and about as large as the bite forces of a vertebrate 20 times heavier [By contrast to this robust empirical evidence for size-related preferences in foraging, the biomechanical factors that underpin it remain poorly understood but see ,28,39). ,39. 28,3ter ants . Peak biHowever, this conclusion is premature, because it remains unclear how the forces required to cut vary with mandible size. For example, one may speculate that cutting forces vary with a characteristic length e.g. ,41,42); ,42; 41,4. 2 (a)A. vollenweideri leaf-cutter ants from two colonies, founded and collected in Uruguay in 2014. The colonies were kept in a climate chamber  at ca 30 cm to the closest fungus box; 25 mm inner tube diameter).We sampled n = 46, [To quantify how the force required to cut thin leaf-like sheets varies with mandible size, we collected two sets of ants across the worker size-range, excluding the smallest workers, which typically do not cut leaves . Firn = 46, ,49). In n = 46, ,50. The n = 46, . To ensun = 46, ,49. To mn = 46, . This men = 46, ).n = 39). These workers were likely to have mandibles worn from the repeated cutting of leaves [Second, we collected fully matured workers from the foraging arena (f leaves . Quantif (b)a). Leaf-cutter ants show no preference between left and right mandible when cutting [a).All ants were sacrificed by freezing, weighed to the nearest 0.1 mg , and decapitated using micro-scissors. The head capsules were split in half along the sagittal plane using a scalpel, and only the left head hemisphere was retained a. Leaf-c cutting , and the cutting . We hencb). Numerous empirical metrics for mandibular wear have been proposed in the literature, including variation of mandible length [a priori. We chose a metric that has been demonstrated to correlate significantly with cutting force in closely related Atta cephalotes ants, so enabling a direct comparison [et al. [W, may be defined as:In order to determine a proxy for the degree of mandibular wear, all mandibles were photographed with a camera mounted onto a light microscope, such that their dorsal surface was in focus . This wb), and the pristine tooth length expected for the same body mass was estimated from a regression analysis on measurements of callow mandibles . The wear index could not be extracted for 17 out of 76 mandibles, because relevant parts of the mandible were obstructed by the head capsule.To calculate the wear index, the lengths of the mandible blade and the two most distal teeth were measured from each photograph b, and th (c)c). The sensor was placed above a stainless steal bending beam with a thickness of 0.40 mm, a width of 10.4 mm and a free length of 28.7 mm, such that the sensor tip was about 400 μm above the beam surface (see [figure 1c).Mandibular cutting forces were measured with a custom-built setup based on a fibre-optic displacement sensor .The sensor was calibrated with ten calibration weights ranging between 10 and 245 mN , covering the range of observed total cutting forces (19–172 mN). Weights were suspended from the mount in increasing order, and at the lever arm at which cutting forces were applied. For each calibration weight, we averaged the sensor output across 5 s after initial force fluctuations had faded . To account for sensor drift, the sensor output was extracted for the unloaded beam at the beginning and end of the measurement, and a linear drift correction was implemented; sensor drift, however, was generally small,  (d)Previous studies on mandibular cutting forces used leaf lamina and floral petals as cutting substrates ,27. ThisPDMS sheets were made with a 4 : 1 (silicon base : curing agent) mixing ratio . The mixed but uncured PDMS was sandwiched between two silanized glass plates, separated by feeler gauges , and pre-cured in an oven at her 48 h . Sheet t−1 (motor speed divided by sample height) to approximate quasi-static loading conditions. The critical displacement to rupture was then extracted from the notched sample based on a time-synchronized video recording. The force–distance curve of the unnotched sample was integrated from zero to this critical displacement to obtain the work done by the applied load. Fracture toughness was then calculated as this work divided by sample width and thickness . Two rectangular samples from each of the eight PDMS sheets were cut and used for pure shear tearing; in one of the two paired samples, a notch of 3 mm length was introduced at the centre of the short side . Both samples were tested at a low strain rate of 0.0067 sils, see ), yieldison, see ).−1 motor speed were conducted with two ‘dog-bone’ samples cut from each of eight sheets according to ISO37 and ISO5893. The Young’s modulus was extracted from the loading region of the stress–strain curve via linear regression between 0 and 10% strain [E = 4.1 ± 0.3 MPa, in good agreement with published estimates [Next, uniaxial tension tests at 0.5 mm s% strain ,70; on astimates . (e)n = 85; Manipulator MM 33, Märzhäuser Wetzlar, Wetzlar, Germany). The mandibles were then positioned using a top-down microscope, such that the dorsoventral head axis was approximately horizontal, the mandibular teeth were roughly perpendicular to the PDMS sheet, and the most distal tooth tip just about extended over the sheet edge (see [c).Individual ant heads were fixed onto a pin holder, which was connected to a 3D micromanipulator (dge (see  and figuc); this procedure ensured that the clamping conditions were kept approximately constant across measurements.We cut a small wedge into all PDMS sheets . We extracted the drift-corrected steady-state total cutting force averaged across 2 mm following the initial peak; the corresponding spacing forces were extracted from the second run at the same motor positions, and averaged across the same distances (d).The beam mount was then moved toward the mandible until the tip of the pre-cut wedge was about to contact the mandibular cutting edge. The sensor recording was started, and the beam mount was moved vertically against the mandible blade, resulting in cutting motion somewhat akin to the ‘blade-like’ cutting behaviour observed in freely cutting leaf-cutter ants ,27. The 0 mm s−1 ,34,72), 0 mm s−1 ,42,73,74istances d.−1 motor speed, using the mandible of a single forager with a body mass of 19.9 mg. Three repetitions were completed per speed, without remounting the mandible between measurements, to reduce confounding effects due to small variations in mandible blade orientation. This variation was also quantified, by measuring cutting forces of one small (5.4 mg) and one large forager mandible (38.4 mg) at a constant cutting speed of 0.3 mm s−1. Both samples were mounted three times onto the pin holder, and cutting experiments were performed three times per mount.Cutting speeds typically vary with forager size; larger ants cut more quickly ,33,34. TF2,6 = 4.43, p = 0.07; ANOVA, large worker: F2,6 = 0.26, p = 0.71); we hence pooled the nine measurements per mandible and calculated the coefficients of variation, CVsmall = 0.10 and CVlarge = 0.03 . The relative force variation was significantly larger for the smaller mandible . We thus performed only a single measurement per specimen, unless otherwise indicated.Mounting had no significant effect on total cutting force , small worker: biological ant mandibles and synthetic PDMS sheets, we performed two additional experiments. First, we measured cutting forces of pristine scalpel blades , positioned such that the blade tip just about extended over the sheet edge to reduce the contact area with the PDMS sheet (n = 5). Second, we performed cutting experiments with mandibles on a biological substrate, the leaf lamina of Japanese laurel, Aucuba japonica; the colonies were regularly fed with these leaves, and the lamina appeared comparatively homogeneous. Laurel leaves were cut from the plant on the day of the experiment, and kept hydrated using wet tissues between collection and measurement. To reduce variation due to material inhomogeneities, we cut all laurel samples from the same plant, from a leaf region close to the mid-vein. Prior to the cutting experiment, we measured lamina thickness and mounted the samples such that the cut ran perpendicular to the mid-vein. We used mandibles of 13 out of the 85 prepared ants, seven foragers (body mass 5.4–38.8 mg) and six callows (body mass 6.2–46.4 mg), mounted once with one to three repetitions per specimen. To account for differences in lamina thickness, tl, we corrected the measured total cutting force, Fm,c, as To contextualize our results based on Across all experiments, measurements were considered invalid and thus repeated when at least one of the following criteria was met: (i) the head capsule came into contact with the clamp or the cutting substrates; this occurred when the head capsule was initially close to the clamp and the PDMS sheet buckled; (ii) the mandible slipped out of the cut; (iii) the steady-state phase was too short to extract a meaningful cutting force (less than 2 mm); (iv) the epoxy fixation of the joint failed, leading to mandible rotation ; and (v) the sample slipped out of the pin holder, as observed occasionally for measurements involving high cutting force (see below). (f)We excluded a total of four out of 46 callows and five out of 39 foragers, because optical inspection of the mandible suggested that small amounts of epoxy contaminated the mandibular teeth, and cleaning attempts failed or caused visible damage. Additionally, we excluded one out of seven forager–laurel measurements, because the total cutting force exceeded the calibration range (greater than 245 mN).10-transformed data.Extraction of the average total cutting and spacing force from the raw data was done in Python (v. 3.9.7 ), and al. 3 (a)Fc, were independent of body mass , but depended significantly on the experimental group . These main effects must be interpreted with caution, as the interaction term was significant , suggesc). Notably, the coefficient of variation also differed significantly by about a factor of two , suggesting that both relative and absolute force variation was larger among foragers. The magnitude of total cutting force extracted for both groups was small in comparison with the total cutting force measured with pristine scalpel blades, 105 ± 34 mN, .On average, total cutting forces of forager mandibles exceeded those of callow mandibles by a factor of two, 64 ± 33 versus 31 ± 7 mN, respectively c. NotablFs, were 5 ± 3 mN, with neither significant differences between experimental groups, nor significant size-effects ). Spacing forces of scalpel blades were 12 ± 4 mN, or c; Wilcoxon rank sum test, forager mandibles versus scalpel blades: W = 8, p < 0.001; callow mandibles versus scalpel blades: W = 10, p < 0.001).Across all ant mandibles, spacing forces, Ff = Fc − Fs, essentially mirrored the results obtained for the total cutting force (b). Fracture forces were independent of body mass , but depended significantly on experimental group , with a significant interaction . Within foragers, Ff tended to decrease with size, but this trend was not significant . Within callows, however, Ff now increased significantly with worker size .Because mandible spacing forces were size-invariant, the scaling of fracture forces, ng force b. Fractua); this difference was not significant . After subtracting spacing forces, however, the difference in fracture force was significant , and averaged 28 mN, similar to the result obtained for PDMS (33 mN).To test if the observed differences in mandibular cutting forces between callow and forager mandibles are also present with biological substrates, we measured cutting forces for a small subset from both experimental groups on laurel leaf lamina. Total cutting forces, corrected for differences in lamina thickness, were 141 ± 44 mN for foragers, exceeding those of callows (95 ± 14 mN) by almost 50 mN a; this d (b)F1,7 = 33.0, p < 0.001) from 29 ± 2 mN at 0.1 mm s−1 to 36 ± 1 mN at 0.3 mm s−1 . Total cutting forces thus increased by only 20% for a threefold increase in speed.The cutting speeds during natural foraging typically vary with both worker size and leaf mechanical properties; larger ants cut faster than smaller ants, and ‘tougher’ leaves are cut more slowly than ‘tender’ leaves ,33,34. W (c)V = 272, p < 0.001), and independent of body mass . This size-independence suggests that absolute wear was the same across sizes, and thus that smaller ants lost a larger fraction of their teeth to wear. Although the relative mandibular wear index, normalized with the pristine length of the second most distal tooth, indeed slightly decreased with size from about 20% for a 3 mg forager to 5% for a 30 mg forager, this decrease was not significant , p < 0.01, R2 = 0.33; see figure 3c), comparable to the rate of 3.7 mN μm−1 reported for closely related A. cephalotes [The mean mandibular wear index of foragers was 8 ± 10 μm, significantly different from zero, which defines the pristine state ,62–659,6Second, we acknowledge that even if experiments with PDMS can provide insights into cutting forces expected for leaves, our cutting experiments do not fully mirror the complexity of cutting behaviour of leaf-cutter ants. For example: mandibles rotate instead of translate; neck muscles may be used to change head and mandible orientation during cutting, and perhaps even directly contribute to cut propagation; and the section of the mandible blade used for cutting may be adjusted to account for local differences in mandible ‘sharpness’, or to dynamically alter the effective mechanical advantage of the mandible lever system. Despite these differences, two arguments suggest that our experiments are informative: cutting forces of pristine mandibles were close to a theoretical minimum for PDMS; and although more complex mandible motion may decrease cutting forces in some cases ,85, out- (a)A. vollenweideri, which grow in almost direct proportion to body mass, Fb ∝ m0.9 [Fc/Fb ∝ m−0.9—a factor of 300.9 ≈ 20 across the size range considered in this study. For materials that could in principle be cut by workers across the size range, the differential scaling of bite and cutting forces affords considerable behavioural flexibility to larger workers, bound by two extreme scenarios.The weak size-dependence of cutting forces stands in stark contrast to the strong positive allometry of maximum bite forces in b ∝ m0.9 . As a reFb/Fc directly determines the maximum possible strain rate of the mandible closer muscle during cutting [First, larger workers may choose to bite with maximum force, i.e. fully activate their closer muscles during cutting. The excess force  cutting ; larger g [Ff∝vc –90. Howeg [Ff∝vc ,75). Indtearing 2 of leaf area per year [ca 3 km of cutting [6)/(12 × 106) = 3, or about 500 times their body length . Such e forager . Consistphalotes . The absphalotes ,40. In ln = 14), equivalent to the difference in maximum bite force between two workers that differ in mass by about a factor of 71/0.9 ≈ 8 [et al. 2023, in preparation). In support of these hypotheses, three lines of evidence may be presented. First, in large workers , cutting forces varied only by a factor of 3 across pristine and forager mandibles, as opposed to a factor of 7 for the mandibles of small workers . Second, although the scaling coefficients for total cutting forces of both callows and forager mandibles were not significantly different from zero . Second, the increase in cutting force with wear is material-independent, and equal to the regression slope extracted for PDMS (2.09 mN μm−1). For mandibles subjected to considerable wear, W = 20 μm, the minimum required cutting forces would thus be shifted up by ca 40 mN for all leaves. For a 30 mg worker, the fraction of cuttable leaves would be virtually unaffected (99.5%), whereas a 3 mg worker would now be able to cut fewer than 10% of tropical leaves, compared to almost 50% with pristine mandibles.Based on the significant increase of cutting forces associated with mandible usage, we may speculate about the downstream effects of wear on the foraging performance of both small and large workers. Previous analysis of leaf mechanical properties, in combination with bite force experiments, suggested that a 30 mg worker may be able to cut almost all species of tropical leaves, whereas a 3 mg worker may only be able to cut about half of them ,40. AlthThe significant increase of cutting forces with wear, and the conjectured reduction in cuttable plant leaves, likely necessitates behavioural adaptations, and may partially explain ‘age polyethism’, i.e. systematic changes in task preferences with worker age. Indeed, leaf-cutter ants with worn mandibles cut at significantly lower speeds, and are more likely to carry rather than cut ; the old (c)The size-invariance of cutting forces and their strong sensitivity to wear have biological consequences. From a mechanical perspective, both results may be surprising at first glance, and thus call for a more thorough evaluation. Intuitively, it appears reasonable to expect that mandibles of larger workers require a larger force to cut the same material. Indeed, the force required to fracture thin or thick model ‘targets’ with biological puncture tools increases significantly with characteristic dimensions of the tool, such as the tip diameter ,14. The UA, and the work that provides this energy has to be supplied by the externally applied load, so that, from a simple virtual work argument, dUext = dUA. Thus, and without loss of generality, the force F required to cut a slab of thickness t is bound from below by F ∼ Gct, where Gc is the energy per unit area of new surface, a characteristic material property [Gc ≈ 100 J m−2 and t ≈ 200 μm (see Methods), so that F ≈ 20 mN. This simple argument lends itself to a definition of an intuitive, quantitative and functionally relevant index for sharpness, S: the required cutting force is equal to the minimum possible force, and independent of tool geometry, if and if only the dimensionless group S = Gct F−1 is unity; the cutting tool may then be considered ideally sharp , suggesting that a further reduction in cutting force through changes in mandible morphology may not be possible. Thus, pristine mandibles of small workers appear ideally sharp, S ≈ 1, at least for PDMS . By contrast, pristine mandibles of larger workers, scalpel blades and the most worn mandibles of foragers have a functional sharpness index S between 2/3 and 1/5; in other words, cutting (and fracture) forces are between 50 and 500% larger than the theoretical minimum, hinting at contributions from cutting-tool geometry. The next task is thus to rationalize the putative influence of mandible geometry on cutting force.In order to rationalize our experimental results qualitatively and quantitatively, we first note that even an arbitrarily sharp mandible will not cut with arbitrarily small force. Cutting is akin to fracture, in the sense that it results in the creation of new surface area. Each unit area of new surface incurs an energy cost dproperty ,71,102. ion, see ). The frUext − dUloss = dUcut [figure 3b [R ; but ot[R e.g. ,103–105)–105Uext gy e.g. ,106–108)–108Uext C = (1 + μ), where μ is the coefficient of friction [σc with the ultimate tensile strength of PDMS . The simple definition of sharpness suggested in equation , and asa [μ ≈ 1 ,110. Thege e.g. ,68,98), ,98, C =  ≈ 17 μm . Pristin ≈ 17 μm , so thatfor PDMS b. The siequation  thus hasR ∝ m1/3 for R may fall anywhere in between. This result may be put to use in two ways.From this cursory analysis, we may surmise that fracture forces are effectively independent of mandible geometry if cRGc−1≫1 ,103. TheR is isometric, i.e. R ∝ m1/3. Plausible alternative hypotheses may be derived. For example, the tip radii of some insect claws depart from isometry and scale as R ∝ m1/2, presumably to ensure that tip stresses remain size-invariant [R from the cutting force measured for a pristine mandible of the largest workers (40 mg in body mass), via equations (4.1) and (4.2), yielding R40 ≈ 5 μm. Next, we use this result to extract a proxy for the proportionality constant a, invoking the null hypothesis of isometry, R = am1/3, and then predict the variation of cutting force across the callow size range from 2.1 to 46.4 mg, using equation . Thus, a difference in cutting edge radius that would increase cutting forces in PDMS by about 40% increases those for the median leaf by a mere 10%. Although the simple model based on dimensional arguments appears to quantitatively capture salient features of our experimental data, more thorough experimental validation, including cutting measurements with a range of natural materials and direct measurements of cutting edge radii, are in order.Second, equation  can be p species , and agaequation  dominateequation  predicts results a. Thus, S = 0.5, corresponding to a doubling of the required cutting force, occurs for R ≈ 12.5 μm in PDMS; the equivalent radius for the median tropical leaf is R ≈ 67 μm—about five times larger. However, there is robust evidence that wear affects leaf-cutter ant performance even when cutting natural materials: the average fracture force required to cut laurel leaf lamina with forager mandibles was about 30 mN higher than for callow mandibles (a), and similar results were reported by Schofield et al. [A. cephalotes workers and Prunus lusitanica leaves; leaf-cutter ants with worn mandibles cut at significantly lower speeds ([S ≈ 1. For example, for R = 1 μm, S = 0.99 ≈ 1, and even for R = 10 μm, S = 0.87, still within 15% of the maximum sharpness for cutting the median leaf. Thus, selection pressure on material properties and edge geometry for the cutting tools of small animals may be less strong than previously suggested [The putatively weak size-dependence of mandible cutting forces for natural materials has two consequences worthy of brief discussion. First, it implies that mandible wear needs to be more severe in order to have an appreciable effect on cutting forces. As an illustrative example, andibles a, and sid et al.  for A. c speeds (, see als speeds ( for simi speeds (,50,94. Cuggested .. 5The ability to cut leaves involves complex interactions between worker size, bite force capacity, wear-dependent cutting forces, plant-material properties and adaptive cutting behaviour. We tried to untangle this complexity, by removing the confounding effects of material heterogeneity and nonlinear mandible motion, and studied the effects of mandible size across two experimental groups with varying levels of mandibular wear.Although smaller ants may experience a larger increase in cutting force from pristine to worn mandibles, cutting forces were nevertheless approximately size-independent, in contrast to our initial hypothesis. The ability to cut leaves is thus mostly affected by size-dependent bite forces, plant-material properties and mandibular wear. As a result, larger ants only need to apply a substantially smaller fraction of their maximum bite force to cut the same material. In agreement with our second hypothesis, the effects of wear on cutting force can be substantial, which may strongly reduce the range of accessible plant tissues for small workers.Pristine mandibles of callow workers are exceedingly ‘sharp’, and even mandibles with moderate levels of mandibular wear cut with similar forces to the ‘sharpest’ pristine scalpel blade; these results indicate morphological adaptations of leaf-cutter ant mandibles to the high mechanical demands of cutting ,45.A natural extension to this work will be to use other materials as cutting substrate, and to test quantitative predictions on cutting force variation and cutting edge geometry for a broader selection of biologically relevant substrates. A careful inspection of the mandibular cutting blade, in combination with mandible abrasion experiments, may reveal structural and chemical adaptations to increase mandibular wear resistance ,45.We hope that the findings of this study will help to increase our understanding of size-specific foraging preferences in leaf-cutter ants, and more generally, may provide a framework to investigate the relative importance of tool geometry versus material properties in biological cutting.


The third most often diagnosed disease globally and the second most prevalent cause of cancer-related death is colorectal cancer (CRC). Numerous human malignancies have been identified to have high expression of ADORA2A. However, it is still ambiguous about its function in CRC. RNA-seq with stable transfected SETDB1 knockdown cells was used to identify differentially expressed genes. Further, knockdown of ADORA2A in CRC cell lines SW620 and HCT116 was performed with siRNA and over expression of ADORA2A in SW480 cells was conducted with plasmids. CCK8, colony formation, wound healing, and transwell assay were used to detect the effects of cell proliferation, migration, and invasion after knockdown and over expression of ADORA2A. Also, apoptosis was analyzed by flow cytometry, apoptosis-related proteins and key PI3K/AKT pathway proteins were detected using Western blotting. ADORA2A was identified after RNA-seq analysis and played an important role in CRC prognosis. ADORA2A was relatively high in SW620 and HCT116 cell lines compared to SW480 cell lines. ADORA2A knockdown in SW620 and HCT116 inhibited cell proliferation, migration, and invasion, while ADORA2A overexpression had the opposite effect. In addition, ADORA2A also impacted the expression of apoptosis-related proteins, including Bcl-2, Bax, Cleaved caspase-3 and Cleaved caspase-9, and reduced apoptosis. Furthermore, this process may include the PI3K/AKT signaling pathway. ADORA2A promotes CRC progression and inhibits apoptosis by the PI3K/AKT signaling pathway. It may contribute to the management and treatment of CRC. Radiation, immunotherapy, palliative chemotherapy, targeted therapy, major surgery, and local ablative therapies for metastases are some of the therapeutic alternatives for CRC that have emerged recently3. However, different clinical and pathological characteristics of CRC might contribute to a variety of prognoses and likely account, at least in part, for therapy resistance5. Therefore, it is crucial to develop new treatment plans and find new molecular markers for CRC prognosis.In 2020, there were 1.9 million new cases of colorectal cancer throughout the world, making it the second most lethal disease6. ADORA2A and DRD1/DRD2 increase the permeability of the blood–brain barrier by inhibiting adenylyl cyclase activity, change the level of dopamine in the brain, and finally cause tremor in Parkinson’s disease7. Studies have shown that ADORA2A is highly expressed in Alzheimer’s disease, and ADORA2A is an independent risk factor for Alzheimer’s disease8. ADORA2A gene polymorphism may affect the response to methotrexate therapy and may serve as a potential biomarker for methotrexate treatment in rheumatoid arthritis9. In addition, evidence has shown in recent years that ADORA2A expression is intimately linked to the development and spread of tumors. The World Health Organization (WHO) grade III gliomas in particular showed high levels of ADORA2A expression10. Compared to non-tumor gastric tissues and cell lines, ADORA2A expression was higher in gastric carcinoma (GC) tissues and cell lines. In addition, ADO interacts with ADORA2A to increase GC cell stemness and ADORA2A is a possible target for enhancing GC radiosensitivity11. ADORA2A is highly expressed in a low-risk group of pancreatic cancer, which provides a new way for immunotherapy of pancreatic cancer12. ADORA2A blockage immunotherapy for renal cell carcinoma that is resistant to treatment13. However, the function of ADORA2A in CRC remains unidentified.ADORA2A encodes the G protein-coupled adenosine receptor known as Adenosine Receptor Subtype A2a (ADORA2A). ADORA2A contact with Adenosine limits anti-tumor immunity by suppressing multiple immune subsets including T cellsIn this study, we identified ADORA2A enriched in CRC progression-related pathways. Higher ADORA2A expression in CRC patients predicted poor survival. And we further explored ADORA2A expression in colorectal cancer cell lines. We found that ADORA2A knockdown decreased cell proliferation, migration, and invasion, and promoted apoptosis. Overexpression of ADORA2A had the opposite results. And the PI3K/AKT signaling pathway may be involved in the process. Conclusion ADORA2A may be a therapeutic and prognostic target of CRC.14. Plus, ADORA2A was relatively enriched in the biological functions of the cAMP signaling pathway and negatively regulated cysteine-type endopeptidase activity involved in apoptotic processes  were identified between sh-SETDB1-HCT116 and sh-NC-HCT116 cells  and intrinsic  pathways21. The last pathway to apoptosis involves proteins from the caspase family, which are also necessary to cause apoptosis. Caspase-3 is the main apoptotic protease, and when it is active, downstream apoptosis cannot help but be generated22. Caspase-9, as an essential initiator, caspase required for apoptosis signaling, is activated on the apoptosome complex23. Numerous studies have demonstrated a strong association between Bcl-2 and Bax levels in CRC. Most Bcl-2 family members have altered expression patterns in CRC tumors, which contributes to disease development and resistance to treatment25. In this work, we discovered that decreased ADORA2A expression raised the levels of Bax, cleaved caspase-9, and cleaved caspase-3 expression in SW620 and HCT116 cells, but decreased Bcl2 expression when compared to the control group. Additionally, overexpressing ADORA2A in SW480 cells decreased apoptosis. The outcomes showed that ADORA2A can cause apoptosis of CRC cells.Apoptosis is a crucial step in eliminating the majority of tumor-causing cells for tissue homeostasis26. AKT, sometimes referred to as protein kinase B, is the primary downstream PI3K signaling molecule. AKT that has been activated then controls cell division, differentiation, migration, and glucose metabolism by phosphorylating its downstream target proteins28. Studies have shown that the PI3K/AKT pathway is crucial for the development of breast, lung, colorectal, and other cancers31. Previous studies proved that inhibition of the PI3K/AKT pathway is of great significance for cancer treatment34. Our research shows that when ADORA2A expression was downregulated in SW620 and HCT116 cells, p-PI3K and p-AKT levels decreased. ADORA2A overexpression in SW480 cells may also promote proliferative, migratory, and invasive traits by upregulating p-PI3K and p-AKT expression, among other malignant features. These findings suggest that ADORA2A may be involved in the control of the PI3K/AKT pathway. However, ADORA2A expression in patients and its relationship to proliferation and apoptosis markers require further verification.The PI3K/AKT signaling pathway is one of the major signal transduction pathways that promotes cell proliferation and inhibits apoptosisOverall, it can be said that ADORA2A was very important for the growth, invasion, and apoptosis of CRC cells. Furthermore, the PI3K/AKT signaling pathway could play a role in this process. A promising new target for CRC treatment may be ADORA2A.32. As more DEGs were fund in SETDB1 kncokdown cells. In this study, we focus primarily on RNA-seq data with SETDB1 knockdown.Previous study, we performed RNA-seq with SETDB1 stable knockdown and overexpression cell line HCT116 by Biomarker Technology Corporation  with log2|fold change|≥ 1.5 and P-value < 0.05http://www.proteinatlas.org) is a database that maps tissues and organs using information on proteins, transcriptomes, and system biology. HPA pathology database patient records for CRC patients were used for survival analysis.The Human Protein Atlas . ADORA2A siRNA was designed by Ruibo  and overexpressed plastic was obtained from OBiO Technology .Human CRC cell lines SW620, HCT116, SW480 used in this study were self-stored in the Department of Pathology, Affiliated Hospital of Southwest Medical University. The origin and cultural conditions were described before−∆∆Ct technique. Primers used were as follows: ADORA2A sense: 5′-TCGCCATTGACCGCTACATTGC-3′, antisense: 5′-AAACGACAGCACCCAGCAGATG-3’, β-actin sense: 5′-CATGTACGTTGCTATCCAGGC-3′, antisense: 5′-CTCCTTAATGTCACGCACGAT-3′.Total RNA was extracted from cells after 24 h of transfection using the total RNA simple kit , and then reverse transcribed to cDNA by the Prime Script RT reagent kit with gDNA Eraser . TB Green Premix Ex Taq™  was used to express ADORA2A mRNA. After normalization to β-actin, the relative quantification of ADORA2A was calculated using the2For cells that had been transfected for 48 h, ice-cold RIPA buffer  containing protease inhibitors and phosphatase inhibitors was used to lyse the cells. . The primary antibodies used in this study were as follow: anti-β-actin ; anti-ADORA2A ; anti-Bcl-2 ; anti-Bax ; anti-caspase3 ; anti-caspase9 ; anti-p-PI3K ; anti-PI3K ; anti-p-AKT ; anti-AKT . The membrane or the captured image was cropped to remove irrelevant parts and present only the proteins of interest. The cropped image had clear borders or lines to show where the original image was cut. The cropping did not alter or misrepresent the original data, affect the interpretation or quantification of the results, or conceal or manipulate any data. The original, full-length images were kept and documented, and were uploaded in Supplementary Information file. The grayscale of the proteins was measured by the software Image J .CCK8 assay was performed with CCK-8 kit . 2000 ~ 2500 cells were transfected for 24 h and placed in a 96-well plate. 100 μl spent medium was replaced with an equivalent volume of new medium containing 10 μl of CCK8 after the cells had been cultivated. The cells were then incubated for 1 h at 37 °C. Using a microplate spectrophotometer, the OD value at 450 nm was measured to calculate the rate of cell growth.2. The clones were stained for 10 min with 1% crystal violet staining solution after being fixed with 4% methanol. An optical microscope was used to assess the number of clones generated.In order to see cell clones, 800 cells were put into a 6 well culture plate and grown for 2 weeks at 37 °C in 5% CO5 cells were transfected and put onto a 6 well culture plate after 48 h. When the cell density is around 90%, cell scratches are conducted using a 10 μl pipet tip, and the cell debris is entirely removed using RPMI 1640 media. Wells were filled with complete media containing 10% fetal bovine serum and cells were incubated at 37 °C in 5% CO2. At 0 h, 24 h, and 48 h, respectively, the microscope camera captured the reduced distance of cell scratches.Approximately 5 × 10Cells (1 × 106) that transfected for 24 h, were seeded in the upper chamber of transwell chamber . 45ul mixed Matrigel (Serum-free medium: Matrigel = 9:1) was added to the transwell chamber . Transfection after 48 h, the upper chambers of the wells contained 200uL of cell suspension, while the bottom chambers received 600ul of RPMI1640 medium with 30% fetal bovine serum. Before being preserved in methanol for 30 min and staining for 5 min with a 1% crystal violet staining solution, the chambers were cultured for 48 h. Using a microscope camera, cells that migrated to the bottom surface of the membrane were counted and recorded.Knockdown cell apoptosis was analyzed using Annexin V-FITC Apoptosis Detection Kit  and overexpress cell apoptosis was analyzed using Annexin V-Alexa Fluor 647/PI Apoptosis Detection Kit . After transfection for 24 h, the cells were harvested and operated according to instructions. Statistical analysis was performed using Flowjo software .Experimental data were analyzed using GraphPad Prism 9.0 software, and statistical analyses were performed using SPSS 26.0. Mean and standard deviation were used to express measured data (SD). The two groups were compared using the Student-Test, and three or more groups were compared using a one-way ANOVA. Statistical significance was determined to be P < 0.05, and the significance shown in *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.Supplementary Information.


Experimental studies have shown that narratives can be effective persuasive tools in addressing vaccine hesitancy, including regarding the vaccine against the human papillomavirus (HPV), which is transmitted via sexual contact and can cause cervical cancer. This paper presents an analysis of a thread from the online parenting forum Mumsnet Talk where an initially undecided Original Poster is persuaded to vaccinate their child against HPV by a respondent’s narrative of cervical cancer that they describe as difficult to share. This paper considers this particular narrative alongside all other narratives that precede the decision announced on the Mumsnet thread. It shows how producing pro-vaccination narratives about HPV involves challenges regarding ‘tellability’ – what makes the events in a narrative reportable or worth telling. We suggest that this has implications for the context-dependent nature of tellability, the role of parenting forums in vaccination-related discussions, and narrative-based communication about vaccinations more generally. Storytelling is recognised as a central tool for making sense of and sharing personal and collective experiences. It can be used to perform a range of functions in different contexts, including moral teaching, community-building, escapism and persuasion. The role that narratives can play in healthcare and the experience of illness in particular is also well documented . This inIn this paper we are concerned with narratives of personal experience told on a thread from the online parenting forum Mumsnet Talk in response to an Original Post in which a parent expresses indecision about vaccinating their daughter against the human papillomavirus (HPV). This thread is an unusually explicit, naturally-occurring example of the persuasive potential of narratives: the undecided Original Poster eventually declares to have been persuaded to consent to their child’s vaccination by one particular pro-vaccination narrative about cervical cancer. The narrative in question, however, was described by the person who posted it as difficult to share.Inspired by this description of the persuading narrative, we explore all 120 narratives posted on the thread prior to the Original Poster’s announcement of a decision. We show how producing pro-vaccination narratives about HPV poses challenges in terms of an aspect of narratives known as ‘tellability’ – what makes the events in a narrative reportable in a particular context . More spThe HPV virus is typically transmitted through sexual contact and can cause several conditions, including genital warts and cervical cancer. HPV vaccination is central to the World Health Organization’s strategy to eliminate cervical cancer, and has been introduced in over 100 countries . In the Narratives have been argued to have a greater persuasive potential than non-narrative persuasive strategies  due to the fact that cognitive and emotional involvement with story-worlds and characters may reduce awareness of persuasive intent and limit resistance to persuasion e.g. . In the The experimental literature on narrative persuasion provides some support for Cawkwell and Oshinsky’s suggestion. In a study involving men who have sex with men, While experimental studies on narrative persuasion can manipulate stimulus texts to control for a range of variables, much less is known about naturally-occurring vaccination narratives, particularly in dynamic and organically evolving communicative contexts where storytelling and responses to storytelling are affected by multiple pressures and tensions, such as relevance to previous contributions, community norms, and the management of self-image and of mutual relationships. The online parenting forum Mumsnet Talk, from which our data is drawn, is one such context, and matters greatly for discussions and decisions about (childhood) vaccinations. Between 50% and 85% of people in a wide range of countries report turning to the internet for information about health-related issues . ParentiThe notion of tellability or reportability, originates from sociolinguistic research on oral storytelling, where narrators need to prevent their audience from responding with ‘So what?’ or any reasonable equivalent – the clearest and most embarrassing indication of a storytelling failure . The conTellability is, first and foremost, a property of events as potential narrative material. Some events are inherently more tellable than others. For example, missing a flight after having one’s passport stolen en route to the airport is inherently more tellable than catching a flight as planned without incident. This captures a relatively context-independent aspect of tellability: unexpected or rare events are inherently more tellable than what is predictable or ordinary. Situations involving problems or conflict, including story participants believing or wanting different things , are alsway in which a story is told also makes a difference to how tellable a narrative is perceived to be. Skilled storytellers can weave an engaging narrative out of unpromising material, while even the most reportable events may fall flat when narrated by an awkward storyteller. In the sociolinguistics tradition, the term ‘evaluation’ is used to capture the different linguistic devices that can be used to emphasise the point of a story, including ‘external evaluation’ via explicit statements such as ‘But it was quite an experience’ or ‘internal evaluation’ via intensifiers such as ‘all’ in ‘I knocked him all out in the street’  versus those that make good narrative material. While this lower-end threshold between the non-tellable and the tellable has received most attention in studies of tellability, Topics that have been described as potentially untellable in previous discourse analytic studies include illness , dying , embarraPrevious studies have also shown how the boundaries at both ends of the cline of tellability are not just dependent on context , but also negotiable in context e.g. . In compMore broadly, online discourse communities such as the contributors to particular Mumsnet threads can be seen as complex self-organising systems. These develop over time as a result of the dynamic interactions of internal and external factors operating along multiple timescales . From thIn the rest of this paper, we present our data and describe how we identified and classified narratives in our analysis. After providing an overview of narratives in the data, we focus on the main pro-vaccination storytelling patterns and point out the tellability challenges they pose and how these are addressed and negotiated by contributors to the Mumsnet thread. We conclude with the implications of our findings.General Health and Children’s Health. Mumsnet Talk is associated with an open, straight-talking approach to parenting discussions  and dates from 9th July 2012, shortly after the Gardasil vaccine was approved for use in the UK. The author of the Original Post  in the context where it occurs. There is some debate, however, as to the precise criteria for classifying a stretch of text as a narrative : 1–25. T[dear son] has had it’.From a narratological perspective, From a sociolinguistic perspective, Abstract: an indication that the speaker has a story to tell and/or a brief summary.Orientation: a description of the setting, including time, place, people, situation.Complicating action: a series of clauses that present the events that are the core of the story.Evaluation: devices that indicate the point of the story, that is, why it is worth telling.Resolution: an indication of the final event.Coda: an indication that the story is finished and potentially some general observations on the effects of the event on the narrator.Within this framework, only a complicating action consisting of two temporally ordered clauses is necessary for a stretch of text to be considered a narrative (e.g. ‘I tripped and fell’). The category of Evaluation is particularly relevant to tellability as it captures the linguistic devices that signal the point of a story.More recently, sociolinguistic research on informal oral storytelling has introduced the term ‘small stories’ to capture ‘a whole range of under-represented narrative activities ranging from literally small and fragmented tellings to refusals to tell and deferrals of telling’ . Most re• Non- or multi-linear unfolding events sequenced in further narrative-making, not linear sequencing of past events.• Emphasis on world-making, that is, telling of mundane, ordinary, everyday events, not world-disruption and narration of complications. : 260.Against this background, we analysed all 207 replies to the above OP up to the announcement of the decision for the presence and characteristics of narratives. We operationalised ‘narrative’ as the telling of one or more actions or events involving personal experiences of vaccination, HPV infection, HPV-related health concerns and illness, and other related topics. In our data, narratives are typically told in the first person and involve the author of the post and/or a family member or a friend. Each narrative was further coded for the following aspects of variation:• Complexity, that is, whether they involved a single action/event cf.  or two o• Main plot focus ;• Vaccine stance .For all types of coding, one co-author coded the entire dataset and another co-author coded a 20% sample of the data based on a shared codebook, to establish reliability. After discussion, agreement between the two coders on the presence of a personal narrative in a post was 100%. Agreement for the characteristics of narratives was 97% for Complexity and 88.1% for both plot focus and vaccine stance, which was deemed satisfactory. Overall, 120 narratives were identified in the 207 replies to the OP that precede the announcement of the decision. Other than narratives, the replies contain information relevant to the HPV vaccine (e.g. statistics about amount of protection) and a variety of other material, such as questions and expressions of opinions, e.g.: ‘How can cervical cancer be caught from boys?’ and ‘YABU’ (You are Being Unreasonable).n = 99, 82.5%) involved two or more actions/events, and the largest proportion  had a pro-vaccination stance, with only a small number of anti-vaccination narratives .As shown in 1. My DD hasn’t had it, we talked about the pros and cons and she decided she didn’t want to have it. I have said that if she even begins to think about becoming sexually active, she will need to have the jab, but (hopefully) that won’t be for at least another couple of years.n = 4, 3.3%), but showed how, as pointed out by Vaccine side effects/harms narratives were relatively infrequent  or because they involved intimate and potentially taboo details (Sex narratives and Illness narratives). In the rest of this paper we demonstrate these challenges in tellability in pro-vaccination narratives, and show how the narratives told as the thread develops create a space in which Taylor ultimately feels able to narrate experiences that would be untellable in most other contexts.the problem is that, as with all vaccine damage cases, millions are vaccinated, a tiny minority have complications  and it’s only ever the ones with bad experiences we hear about.Prior to announcing their decision, the Original Poster returns to the thread and makes the following comment:The 120 replies that precede the decision announcement do include 41 Vaccine Uptake narratives, 38 of which are pro-vaccination. Of these, however, 12 (31.6%) take the form of minimal single-action narratives (examples 3 and 4), sometimes including an explanatory or evaluative comment (example 4):dear son] has had it.3. On vaccination my older ds  had hers, after a bit of plea bargaining, she hates needles. We agreed she’d have her HPV and I’d let her off her flu one. She’s on the list for ridiculously mild asthma and reacts really badly to them .5. DD1 [[dear daughter] and I discussed it at length and she decided to get it done. She had been off sick with appendicitis when all the others had it. She decided to get it done and made doctors appointment herself to have it.6. My DD Example (5) mentions the daughter’s attitude to needles as a potential barrier to vaccination and includes an unrealised possibility that could have made the vaccination more eventful than it turned out to be . Several linguistic choices emphasise the point of the story by providing the writer’s evaluation, including the intensifiers ‘ridiculously’, ‘really’ and ‘at all’. Example (6) involves a different barrier to vaccination – an illness that caused the narrator’s daughter to miss out on routine vaccination at school and required her to make a separate appointment. In contrast to (5) the daughter is cast as the heroine who manages to be vaccinated against the odds, thanks to her own initiative. She is twice the grammatical subject of the verb ‘decide’, and evaluative devices emphasise her involvement in the discussion (‘at length’) and her pro-active approach in arranging the vaccination (making the appointment ‘herself’).While the authors of these pro-vaccination narratives have to negotiate the lower threshold of the tellability cline, by contrast, contributors who tell pro-vaccination stories involving sex or illness need to negotiate the upper threshold, between the tellable and the untellable.Our Mumsnet thread includes 33 personal narratives in which the writer or someone close to them experiences HPV infection, abnormal smear test results and/or cervical cancer. Of these, 20 are pro-vaccination, as in the extracts below:7. I didn’t have sex until I was 22, never smoked, was a veggie health freak, two boyfriends, regular smears. And hey presto by 30 I had what they call ‘carcinoma in situ’ and had two big chunks of my cervix cut out. Luckily I was able to have two kids afterwards but ended up with a hysterectomy at 35 as the remaining cells were starting to change.8. I practiced safe sex, had all the usual tests before going on the pill with ex partners and them the same. I still contracted the HPV virus and I’ve had abnormal smear tests for the past 5 years. Your daughter could only have one partner her whole life but if that partner is carrying HPV then at some point she’s going to become at risk to it. If you can reduce that risk then imo it’s worth her being vaccinated.Generally speaking, experiences of problems and surprises are more tellable than smooth and predictable happenings. Indeed, in both examples (7) and (8) unexpected HPV-related illness provides compelling and potentially involving narrative material: repeated tests, different stages of illness, and the consequences of the illness for the authors of the post.The particular nature of HPV infection and the context of the thread also lead to the provision of highly personal details about the contributors’ sex lives. As mentioned, the human papillomavirus is primarily transmitted through sexual contact. Changes to the cervix caused by the virus can potentially be detected via smear tests before the development of cervical cancer. Indeed, the author of the Original Post makes a reference to her own ‘safe sex and smear tests’ as potential alternatives to the vaccine for avoiding HPV infection or its consequences. In this context, as shown in examples (7) and (8), the authors of Illness narratives tend to choose to reveal their own safe and/or restrained sexual practices to suggest that these are not sufficient to protect against HPV infection and its potential consequences. In both cases, a contrast between safe sexual behaviour and the contracting of HPV or cancer is linguistically marked as part of the evaluative component of each narrative: ‘hey presto’ in the second sentence of (7) and ‘still’ in the second sentence of (8). Thus, maximising the tellability of one’s experiences of HPV-related illness in the context of the thread leads to the sharing of details that are not normally or easily tellable in other contexts, as they could be perceived as crossing the upper threshold of the tellability cline, between the tellable and the untellable.Similar considerations apply to the 12 narratives in our data where sexual activities are the main plot focus, 10 of which are pro-vaccination. The author of extract (9) below, for example, begins by saying that the Original Poster would make ‘the wrong decision’ by withdrawing consent to their daughter’s vaccination, and then goes on to add:9. I believe in no sex before marriage and my husband and I were both virgins when we married and we’ll be teaching our children  to believe the same, BUT that doesn’t mean they will or that that their future partners will.Here the narrative spans both past and future events and involves both existing and hypothetical individuals to make the point that practising and teaching sexual restraint does not guarantee that one’s children will not need the protection provided by the HPV vaccine. As part of this, the writer includes the highly private detail that she and her husband did not have sex until after marriage.While there is no linear progression in terms of plot focus or other characteristics of the narratives told by contributors, it is nonetheless notable that the ‘clincher’ narrative in From a structural perspective, Taylor’s post includes an abstract (second paragraph), an element of orientation (‘even though I had regular smears’), a complicating action consisting of a series of events (third and fourth paragraphs), and a coda  spelling out the implications of the experience for the writer (‘My life has changed enormously and permanently’) and for the OP and other hesitant readers (‘If vaccination could prevent this happening to other women . . .’). A range of evaluative devices are spread throughout the post, including: intensifying expressions, for example, ‘major’, ‘long term’, ‘enormously’, ‘permanently’; the reference to cervical cancer causing many deaths worldwide, and to the writer being ‘one of the lucky ones’ and ‘genuinely fortunate’ in spite of everything; and the use of simile and metaphor to emphasise the consequences of illness and surgery (‘like being run over by a train’) and the professional achievements undermined by the cancer (‘after working my socks off’). The narrative also provides the perspectives of other people affected by the writer’s illness and subsequent changes , and outlines the kinds of unrealised possibilities or virtual narratives that However, several of the details provided in the narrative would arguably be untellable for most people in most other contexts. The writer discloses bladder problems due to nerve damage, and problems in her sex life due to the removal of part of her vagina. One of the unrealisable possibilities that are outlined in the post is finding another sexual partner if anything happened to the writer’s husband. In addition, as with Sex narratives and other Illness narratives, there is a moral angle to the identities that the writer constructs for herself. She spells out that she developed cancer in spite of keeping up with her smear tests, and emphasises how her cancer has caused a shift from a position of high social worth associated with a hard-earned professional role to being ‘a non working person on benefits’, which is implicitly presented as less worthy and potentially shameful, and therefore a challenge to her self-esteem.In contrast with Vaccine Uptake narratives, therefore, telling pro-vaccination Illness narratives and Sex narratives in our data involves a challenge at the upper threshold of the cline of tellability, between the tellable and the untellable. This challenge is caused by the sexual mode of transmission of the virus, the fact that HPV infection and its consequences mainly  affect the female reproductive organs and, in the Mumsnet thread under discussion, the reference to practising ‘safe’ sex in the OP. The telling of these potentially untellable elements of the narrative is facilitated not only by the anonymity provided by Mumsnet, but also by similar elements being included (with less detail and spread across narratives) in preceding posts. These collaboratively raise the upper threshold of the tellability cline for this specific thread, creating a space in which Taylor’s story can be told, albeit after an explicit negotiation of that upper threshold. As noted earlier, in this particular case, the resulting details and personal exposure are explicitly appreciated by several other contributors, and ‘clinch’ the Original Poster’s stated decision in favour of consenting to vaccination.Previous research has provided both theoretical accounts of and some empirical evidence for the persuasive potential of narratives, including in the context of vaccine hesitancy. This persuasive power has been proven particularly for HPV-related narratives of disease that are told in the first person, involve social barriers to vaccination, focus on protagonists who survive their illness, and incorporate the provision of information . HoweverAt the lower threshold of the cline of tellability , the chaFocussing on other end of the tellability cline, experimental studies showing the persuasive potential of narratives tend to involve experiences of HPV-related illness e.g.  and thesIn some ways, the context of the specific Mumsnet thread we have analysed increases such challenge due to the reference to ‘safe sex’ in the OP, so that contributors offering pro-vaccination Illness and Sex narratives often mention their own sexual restraint to establish their credentials as tellers of pro-vaccination cautionary tales of HPV-related illness. This arguably makes it more likely that the undecided Original Poster will want to identify their daughter with the protagonist, but also potentially promotes problematic and highly gendered standards of sexual restraint for women in particular , and mayIn other ways, however, the context of our specific thread also facilitates the telling of the ‘clincher’ narrative. Although there is no clear sequential progression along the cline of tellability in the narratives posted on the thread, it is nevertheless the case that the clincher narrative is preceded by several shorter Illness narratives that each contain some sensitive and personal elements. This likely makes it easier for the clincher narrative to be told  and highlights the context dependence of (un)tellability. It also points to the importance of anonymous online communities in collectively raising the upper threshold of tellability within a dynamically developing context, and enabling intimate and uncomfortable revelations to be made . This leIn public health messaging about the HPV vaccine the upper threshold of the tellability scale is not as easily negotiable, and the authenticity that is associated with narratives told on online forums such as Mumsnet Talk is hard to replicate. These issues may exacerbate the difficulties with addressing vaccine hesitancy around the only vaccine to date that has been proven to prevent a type of cancer. However, our results do point towards the crucial importance of engagement with vaccine hesitant individuals and communities over longer periods of time , as well as to the vital role of real lived-experience accounts and peer-to-peer interactions in addressing vaccine hesitant attitudes in ways that are appropriate for different communities at different points in time.


Functionally related genes are well known to be often grouped in close vicinity in the genomes, particularly in prokaryotes. Notwithstanding the diverse evolutionary mechanisms leading to this phenomenon, it can be used to predict functions of uncharacterized genes.e.g., pathogenic ones. To this end, an optional procedure for averaging occurrences within subtaxa is available.Here, we provide a simple but robust statistical approach that leverages the vast amounts of genomic data available today. Considering a protein domain as a functional unit, one can explore other functional units (domains) that significantly often occur within the genomic neighborhoods of the queried domain. This analysis can be performed across different taxonomic levels. Provisions can also be made to correct for the uneven sampling of the taxonomic space by genomic sequencing projects that often focus on large numbers of very closely related strains, http://bioinfo.sggw.edu.pl/neighborhood_analysis.Several examples show this approach can provide useful functional predictions for uncharacterized gene families, and how to combine this information with other approaches. The method is made available as a web server at  The term operon refers to a genomic cluster of co-regulated genes in prokaryotes. They encode functionally related proteins that are under the control of a single regulatory signal or promoter. Numbers of highly conserved operons in genomes of bacteria or archaea are usually small, more often the operons are unique and rare   is a staProbably the most advanced and broadly used tool for gene neighborhood analysis is STRING. In its current version (11.5), the database contains data about 14,000 genomes of which nearly 90% are prokaryotic. The STRING neighborhood analysis tool searches for homologs of the query gene(s) in all genomes. The software predicts functional partners based on combined scores from conserved presence in genomic neighborhoods, gene fusion, co-occurrence in genomes, and co-expression, as well as information from functional databases and text mining .The next interesting tool for the analysis of gene neighborhoods is FlaGs. It compares conservation of gene neighborhoods across evolution. For an input set of proteins, it clusters sequences found in their genomic neighborhoods using the sensitive sequence search algorithm, Jackhmmer. For ease of interpretation, it provides a phylogenetic tree of the input proteins annotated by the conserved genomic neighbor clusters .Another tool, SLING, can be used to search for divergent occurrences of functionally related genomic neighborhoods by requiring the presence of a conserved “hit” gene together with predefined “partner” (neighbor) genes .The next three tools take a different approach to neighborhood analysis because they focus on visualization. The Microbes Online portal provides information about over 1,700 prokaryotic genomes. It integrates functional genomic data and comparative genomic analysis providing a structured view for comparative genomics . This sie.g., coloring by COG or Pfam domains)  is a database that makes available information about over 80,000 prokaryotic genomes. This portal does not provide neighborhood analysis, however, it allows to visualize genomic neighborhoods of homologs of query genes with functional annotations (domains) . BioCyc domains) .EFI-GNT is a tool that uses the sequence similarity network (SSN) as an input. SSN is generated by other EFI tools available on their site. This network is created based on found homologs of query protein sequence in bacterial, archaeal or fungal organisms. Similarities and differences from alignments are used to calculate edges between homologs and related proteins are grouped together in clusters. Then, for each sequence in SSN, the algorithm collects domains from the closest neighborhood. Returned data is used to create networks that show the presence of Pfam domains in each SSN and its reversed visualization where is shown in which SSN each domain is present .Currently, the amount of biological data produced thanks to advances in high-throughput technologies is huge. Easy access to data creates opportunities to develop new analysis tools. However, for a productive insight, not only visualization but also appropriate statistical evaluation is necessary . Of all In this article, first, we present the statistical approach for functional analysis of gene neighborhoods. Then, we offer an overview of how much information this approach may provide for the thousands of proteins containing “domains of unknown function” (DUFs). Next, we discuss in more detail functional predictions for several such DUFs, one of which has been recently experimentally characterized. Finally, we compare our method with the popular STRING server.http://bioinfo.sggw.edu.pl/neighborhood_analysis. Code of ProFaNA algorithm without data is stored at https://www.github.com/steven0seagal/ProFaNA (DOI 10.5281/zenodo.7993764).The algorithm, instructions and sample results of the presented tool are available at https://img.jgi.doe.gov/) were downloaded in December 2020. In this database, genomic information on archaea, bacteria, eukaryotes, plasmids, and viruses are stored. The current IMG/M database (as of December 2020) contains 88,754 bacterial genomes  annotations, using mapping data from logy.org . The GO e.g., BLAST or HHblits. Either way, the query is always translated into a set of genomic neighborhoods. The necessary input information includes, besides the queried domain or gene list, the size of the genomic neighborhood to be investigated described as the number of base pairs around the query gene. Typically, the size of a neighborhood set to 5,000 bp in each direction (10 kbp of genomic sequence) corresponds typically to approx. A total of 10 genes in the whole neighborhood. Although we expect most operon-like functional units will not exceed 10 genes, this parameter can be changed by the user. In taxon-restricted analysis, taxa available for neighborhood analysis (genera or families) were chosen by requiring a large number of available genomes. Due to the possibility of a high false discovery rate that can be a serious problem when multiple tests are performed , the user can decide to apply a multiple test correction such as Bonferroni or Benjamini-Hochberg correction  prokaryotic all-genomes neighborhood analysis or (2) prokaryotic taxon-specific neighborhood analysis. In the case of the first approach, either all genomes may be treated equally, or averaging can be performed at selected taxonomic levels: genus, family, order, class, or phylum . The querrection .k or more target domain occurrences in n query neighborhoods by chance, i.e., if the target domain was randomly distributed in the genomes. To this end, we calculate the probability mass function using a modified Poisson distribution (od) and the probability of finding the target domain in a randomly selected genomic region of the size of a neighborhood. k is the observed number of occurrences of the target domain in query domain neighborhoods  of observing k or more occurrences of the target domain by chance, we subtract from unity the sum of probabilities for cases where the number of occurrences k’ is less than k  that consists of many lower-level sub taxa  we conduct neighborhood analysis and collect results within sub taxa  to extract values needed to calculate Poisson distribution for each target domain. Then, we identify the subtaxon with the largest number of query neighborhoods (n-submax). For all the other subtaxa “i”, we calculate the difference between n-submax and n-subi. Then, to assign equal weight to every subtaxon, we randomly draw (n-submax−n-subi) neighborhoods from subtaxon “i” and include them in the analysis. This modification is available as an option in two types of analysis: for taxon-specific analysis—at the family level  and at the all-database level where the user can select whether an averaging method will be used or not. At the family level, 12 families are available, each containing more than 1,000 genomes. In the “all database” analysis, averaging can be performed on five taxonomy levels . At each level, subtaxa containing at least 25 genomes are considered for the averaging analysis. For each of these taxonomy levels, we perform neighborhood analysis for every taxon  that include more than 25 genomes are present in the database).In our method, during the analysis of a higher-level taxonomy group  . Thus, cEscherichia coli K-12. For each query DUF, we use ProFaNA to identify a list of domains significantly often occurring in its neighborhoods. Then, using the STRING database, we identify E. coli proteins that contain the queried domain and collect domains that are identified by the STRING algorithm as functionally related. In the end, we compare the lists of related domains identified by STRING and by ProFaNA. This approach was used for a global comparison of the most abundant DUFs.The main reason to create the ProFaNA tool was to develop an algorithm that will help analyze prokaryotic genomic neighborhoods to guide experiments. For comparison of ProFaNA with STRING, we focused on domains of unknown function (DUFs) present in http://bioinfo.sggw.edu.pl/neighborhood_analysis/. Upon reaching the website, users are given the option to choose between three input formats: Pfam domain ID, list of genes, or protein sequence. Depending on the chosen input format, different forms containing pre-defined parameters open up to facilitate analysis.The ProFaNA server discussed in this article can be accessed through its dedicated website, which can be found at P scores, and an analysis name. If the user chooses the Pfam domain ID as their query input, they must provide the Pfam domain identifier and select a group of organisms to be searched for the presence of the queried domain. The rest of the input fields are the same as those for the gene list option. In addition to the Pfam domain ID and gene list input options, the ProFaNA server also allows users to input protein sequences in FASTA format to search for homologs using a DIAMOND search. Additionally, the E-value threshold for sequence similarity has to be selected. The rest of the fields are the same as those for the gene list option.If the user selects the gene list as their query input, they must provide the server with a list of genes (IMG identifiers), the size of the neighborhood to be analyzed, the multiple test correction method for statistics, a choice of DNA strand for analysis, the cut-off for P scores, and the numbers of occurrences in query neighborhoods and in all analyzed genomes. The “percentage in neighborhoods” column provides information about the fraction of query neighborhoods in which the target domain is found. Information about Gene Ontology (GO) terms for each target domain is also included, if available.Once the analysis is complete, users can expect to receive an email notification with a link to their results. This email notification system helps users stay informed about the progress of their analysis, especially when the calculation time is longer than expected. After receiving the email notification, users can access their results on the ProFaNA website. The results are displayed in a user-friendly table that includes information about target domain Pfam IDs, domain names and brief descriptions, Users can easily search through all values in the output table, and the table can also be downloaded in CSV format for further analysis. Additionally, we provide a taxonomy report that shows in which genes and genomes the query domain was found. This can be useful for users that want to make additional analysis on a smaller taxonomy group of choice.We are aware that IMG Pfam annotation is not up to date, and it is a downside of our tool, however, as soon as IMG/JGI updates its database with up-to-date Pfam domain annotations, we will update our records as well. The process of updating our software database with data from IMG/JGI website involves multiple steps. Firstly, existing and new data are compared and any discrepancies are identified. Next, new data is downloaded and integrated into the ProFaNA database while any deprecated data is removed. Statistics are then prepared for the updated database. The entire process can take up to a week, depending on the size of the database and the amount of new data.P score below 10−5 and present in at least 5% of query neighborhoods). Typically, these significant domains occurred in between five and 60% of neighborhoods. Additionally, from the previous analysis we considered the 100 most common DUF domains in the database and considered the distribution of significant domains  . It was Subsequent experimental studies confirmed most of these predictions. The crystal structure of SelO indeed showed the predicted protein kinase-like fold. Further, it was shown that unlike kinases, SelO transfers AMP instead of phosphate to protein substrates and SelO-mediated AMPylation is involved in redox homeostasis and oxidative stress response .Escherichia, Salmonella, Shigella, Klebsiella, within the family Enterobacteriaceae and in the whole database    and S23.Escherichia genomes, we found 17 domains significantly overrepresented in the neighborhoods. Sixteen of them occur in SelO neighborhoods in more than 85% of Escherichia genomes. Thus, they form a conserved SelO neighborhood typical for the Escherichia genus and may be related functionally to SelO. Additionally, in this group, eight domains do not occur in the analyzed genomes outside SelO neighborhoods , Salmonella (36%) and Klebsiella (14%). In this group, there are six domains that are found only in the proximity of SelO domain: hemP, GSHPx, B5, PPDK_N, Kinase-PPPase, FDX-ACB . These domains form a conserved co-localized group. First, two kinase domains PPDK_N and Kinase-PPPase suggest that the SelO domain function may be related to phosphorylation. The other two domains are GSHPx found in glutathione peroxidase, and hemP responsible for uptake of hemin as source of iron. Last three domains, B3_4, B5, FDX-ACB are found in Phe-tRNA synthetase and it was proven that oxidation of Phe-tRNA synthetase positively regulates mechanisms that control translation process and help cell to properly respond to oxidative stress . To sum P score < 0.00001) in the DUF5565 neighborhoods, 10 were found in at least 5% of neighborhoods.This uncharacterized domain, evolutionarily conserved in animals, is also found in some bacterial genomes. To conduct neighborhood analysis, bacterial homologs were collected from the IMG/JGI database using BLAST search. Thus, 390 homologs were used as input for ProFaNA neighborhood analysis. Among the protein domains significantly overrepresented (P score < 0.00001) in DUF5565 neighborhoods . Then we mapped significant domains to genes and created a gene relationship network. Analogous gene-gene relations were downloaded from the STRING database. Comparing the two networks, a substantial number of relations  are captured by both approaches (E. coli genomes considered and in the details of statistical evaluation of significance of co-occurrence in neighborhoods.First, we considered all Pfam domains present in the best-known proaches . Howeverproaches . DiffereThe ProFaNA tool is, to our knowledge, the only one that allows analyzing prokaryotic genomic neighborhoods on a large scale, applying statistical evaluation, including thousands of genomes. Our organism dataset was made based on the IMG/JGI Portal, currently one of the biggest public microbial genomes databases. Our algorithm can be used to analyze the genomic neighborhoods of protein domains for genera or at higher taxonomic levels, while considering uneven distribution of sequenced organisms by application of an optional averaging procedure. Comparison with the STRING database suggests that ProFaNA can broaden the functional relationship networks. One limitation of the current version is its reliance solely on Pfam domains to characterize neighboring proteins. This tool is designed for scientists who want to explore the genomic neighborhood of a query domain, search for patterns of domain genomic proximities in different taxonomy groups or try to predict domain function based on neighborhood co-occurrence with better-characterized domains.10.7717/peerj.15715/supp-1Supplemental Information 1Escherichia sp., Salmonella sp. and Klebsiella sp. genomes, red circle represents domains from the Enterobacteriaceae family.Comparison of significant domains found in genus-level and family-level analysis. Green circle represents significant domains from results for Click here for additional data file.10.7717/peerj.15715/supp-2Supplemental Information 2Total of 42691 neighborhoods shown. Rows and columns correspond to the SelO neighborhoods, color bar shows value of Jaccard Index, representing similarity in protein domain composition of the neighborhoods. The heatmap is clustered using single linkage clustering.Click here for additional data file.10.7717/peerj.15715/supp-3Supplemental Information 3Total of 4130 neighborhoods shown. Rows and columns correspond to the SelO neighborhoods, color bar shows value of Jaccard Index, representing similarity in protein domain composition of the neighborhoods. The heatmap is clustered using single linkage clustering.Click here for additional data file.10.7717/peerj.15715/supp-4Supplemental Information 4Related to Figure 5. Featured domains are those that occur in genomes in close proximity of SelO but very rarely elsewhere in the genomes.Click here for additional data file.10.7717/peerj.15715/supp-5Supplemental Information 5Click here for additional data file.


Therefore, they play pivotal roles in the PI3K/Akt pathway. Here, we investigate the role of the C2 domain in membrane interactions of PTEN and SHIP2, using molecular dynamics simulations and free energy calculations. It is generally accepted that for PTEN, the C2 domain interacts strongly with anionic lipids and therefore significantly contributes to membrane recruitment. In contrast, for the C2 domain in SHIP2, we previously found much weaker binding affinity for anionic membranes. Our simulations confirm the membrane anchor role of the C2 domain in PTEN, as well as its necessity for the Ptase domain in gaining its productive membrane-binding conformation. In contrast, we identified that the C2 domain in SHIP2 undertakes neither of these roles, which are generally proposed for C2 domains. Our data support a model in which the main role of the C2 domain in SHIP2 is to introduce allosteric interdomain changes that enhance catalytic activity of the Ptase domain.Phosphatase and tensin homologue (PTEN) and SH2-containing inositol 5′-phosphatase 2 (SHIP2) are structurally and functionally similar. They both consist of a phosphatase (Ptase) domain and an adjacent C2 domain, and both proteins dephosphorylate phosphoinositol-triphosphate, PIP This process is part of several signaling cascades [3 promotes proliferation of the cell, so by removing PIP3 from the membrane, PTEN acts as a tumor suppressor, and mutations of the PTEN gene are associated with cancer [C2 domains are structurally conserved modules of about 130 residues, which fold as beta-sheet sandwiches [orms PI3,P2. This SHIP2) bound only weakly to a lipid bilayer mimicking mammalian inner membrane [SHIP2 even dissociated from the membrane, verifying the weak lipid binding. Those preliminary results challenge the conception of C2 as a membrane anchor, at least in the case of SHIP2. In this study, we therefore investigated the role of C2SHIP2 and compared it with the corresponding domain of the related protein PTEN. We used MD simulations to study the protein complexes containing both Ptase and C2 domains (denoted here as full-length PTEN or SHIP2), as well as the isolated Ptase domains. The results can be directly compared with the previous simulations of isolated C2 domains of the two proteins [Both PTEN and SHIP2 consist of an N-terminal phosphatase (Ptase) domain, followed by a C2 domain. The Ptase domain contains the active sites, whereas the C2 is an auxiliary domain and is widely believed to facilitate membrane binding in SHIP2 and PTEN, as well as in several other protein classes . Therefomembrane . In all-proteins , as we uThe full-length PTEN consisted of residues 1–403, whereof the structure of residues 14–281 and residues 312–351 was taken from the X-ray crystal structure (PDB 1D5R) . MissingPTEN consisted of residues 1–186 and C2PTEN of residues 191–352 of the full-length construct.We previously simulated lipid binding of a Ptase-C2 core of PTEN , but thaSHIP2 consisted of residues 418–729 and C2SHIP2 of residues 747–862 of the full-length construct.The full-length SHIP2 was simulated previously  and consmartinize script [insane script [2 (Martini lipid POP2 [www.cgmartini.nl). The lipid headgroup is based on PIPP2 [+ or Cl− ions. For PtaseSHIP2 simulations, the box was 10 × 10 × 20 nm3; for PtasePTEN simulations, the box was 7 × 7 × 18 nm3; and for full-length simulations, the box was 12 × 12 × 24 nm3. The simulations were performed using the Martini 2.2 forcefield [−1; the v-scale thermostat [The initial structures were rotated through a random angle, then centered before they were transformed into a coarse-grained model following the Martini 2.2 coarse-graining scheme , using te script . An elase script . The proe script . The mempid POP2 ) P2 . The insPP2  and neutrcefield . The conrcefield  set at 1ermostat  was set −1. In both equilibration simulations, the protein atoms were position restrained by a force constant of 1000 kJ/mol in all directions. Finally, a 600 ns production run was generated with a 2 fs time step and without position restraint on the protein.The coarse-grained binding poses were converted to all-atom models using CG2AT   was calculated with gmx rotmat. Binding poses were selected from the frequency densities in the zzR vs distance 2D landscapes.The distance between the protein center of mass and the membrane center of mass (after centering of the protein in the box) was calculated with the GROMACS algorithm gmx distance tool and minimum distances by gmx mindist. Root mean square deviations (RMSD) were used to monitor the difference between the binding pose of isolated Ptase and Ptase in the full-length Ptase–C2 complex bound to the same lipid membrane. The RMSD were calculated after alignment in the xy-plane  and around the z-axis using an in-house script. RMSD was also used to monitor the structural change after converting the coarse-grained binding pose to all-atom structure and simulating it in the CHARMM-36 m all-atom forcefield [gmx rms. Finally, RMSD were used to compare the SHIP2 full-length and the PtaseSHIP2 simulation with a previously reported crystal structure, respectively [gmx rmsf. For the same simulations, root mean square fluctuations (RMSFs) were used to monitor fluctuations of regions in the Ptase domain of SHIP2 during the 600 ns simulations. These were likewise calculated with gmx rmsf.The center-of-mass distance was calculated with the rcefield  with TIPectively . For thiz-coordinate, using the GROMACS pull code. A pull rate of 0.2 nm/ns with a harmonic (umbrella-type) constraint with a force constant of 1000 kJ/mol/nm2 was used. The binding pose was likewise pushed 0.24 nm closer to the membrane center, with the same settings, except that the rate was inverted to be −0.2 nm/ns. Frames with a distance of 0.05 nm were taken from the push and pull trajectories, ranging from the frame where the protein was pushed 0.24 nm closer to the membrane center of mass than the binding pose to a frame where the protein was pulled to a distance of 7 nm (for Ptase simulations) or 8.5 nm (for full-length simulations) from the membrane center of mass. Position restraints were applied on all lipids in the pull simulations (not in the push simulations or umbrella simulations) to prevent lipids being pulled out of the membrane together with the protein. Each frame (window) was sampled for 1 μs with a time step of 35 fs, without position restraint on the lipids. The potential of mean force (PMF) was calculated from the forces using the WHAM algorithm [gmx wham [Binding poses from the coarse-grained simulations were taken as initial structures. These were then pulled away from the membrane along the lgorithm , as implgmx trjconv was used for extraction. Resolvation was performed with insane using the same box size as the box from which the protein was extracted. Topology files were changed manually to accommodate the changes. The system was minimized and equilibrated in the NPT ensemble for 10 ns, with a time step of 30 fs and with the protein restrained by a 1000 kJ/mol/nm2 force constant. Umbrella sampling was then performed as described above.We wished to obtain the free energy contributions for C2 and Ptase in the full-length binding mode, respectively. To this end, Ptase and C2 from full-length PTEN were extracted, and each domain was then placed back by the membrane and resolvated. The same was carried out for Ptase and C2 from SHIP2. The GROMACS function Free energy surfaces were generated with PyMOL (vacuum electrostatics). Structures were visualized using PyMOL , and plots were generated in Python3, using Numpy  and Matphttps://github.com/andreashlarsen/John2023-PTEN_SHIP2 (accessed on 23 March 2023).Scripts generated for running the simulations are made available at GitHub: PTEN, which was not modeled in the PTEN structure in ref [zzR. zzR is the zz-component of the rotation matrix with respect to a reference structure, in this case, the last frame of the first repeat. Thus, zzR represents a difference in binding orientation. Two binding modes were observed . This adds up to 212 kJ/mol, i.e., 30% more than the estimated free energy of the binding of full-length PTEN, which was 163 kJ/mol. Correspondingly, for SHIP2, the sum of the free energies from the components was 105 kJ/mol, which is 9% more than the estimated free energy of the binding of full-length SHIP2, which was 96 kJ/mol. The higher degree of freedom of movement for the single domains (which are not constrained by neighboring domain) may lead to this difference through higher entropy and more optimal alignment with the membrane.The energy contribution from PtasePTEN has a positive groove (dominated by Arg and Lys) facing the negatively charged membrane  facing the membrane, which prevent tight binding to the anionic membrane  and only one anionic one (Asp) (HKQNKMLKKDKM), and loop78 contains four cationic residues (Lys and Arg) against two anionic residues (Asp) (KNDLDKANRYFSPNFK). Although loop12 contains no cationic residues and one anionic residue (Glu) (FETIPMFSGGTCN), the overall charge of the three loops of C2PTEN is highly positive (+5e) and provides an attractive binding interface for anionic lipids, such as PIP2. In C2SHIP2, loop12 is cationic (KTASRTK), but loop 56 (KSMDGYESY) and loop78 (HRGEE) are slightly anionic, and the total charge of the three loops C2SHIP2 is only +1e.The C2 domains of full-length PTEN and full-length SHIP2 have the same overall binding orientation with respect to the lipid membrane, with loop12 (the loop between helix 1 and helix 2), loop56, and loop78 facing the membrane and the N- and C-termini pointing away from the membrane. Loop56 of C2The same overall binding orientation was observed for isolated C2 domains of PTEN and SHIP2 , and this binding mode is typical for calcium-independent Type II C2 domains .2s in the membrane stayed constant  and helices α5-7 (residues 615–643) of PtaseSHIP2 [SHIP2 (SHIP2 (residues 587–594), which is in the interface between the domains, is less flexible in the presence of C2SHIP2, as shown from a decrease in RMSF (SHIP2 does not change fluctuations of loop L4 and helices α5-7 significantly (SHIP2 (residues 531–539) showed a strong reduction in fluctuations in the presence of C2SHIP2 . Previous MD simulations showed that C2aseSHIP2 . These s2 [SHIP2 A. We als in RMSF B. Howeveficantly B, althou C2SHIP2 B in our In order to probe the different roles of C2 in the phosphatases PTEN and SHIP2, we measured their contributions to the free energy of lipid binding . In PTENPTEN and C2SHIP2 under the same conditions as in the present study, but with a smaller membrane (7 nm × 7 nm versus 12 nm × 12 nm) [PTEN was estimated to 78 ± 4 kJ/mol (compared with 109 ± 1 kJ/mol with the larger membrane), and the binding energy of C2SHIP2 was estimated to 23 ± 1 kJ/mol (compared with 30 ± 1 kJ/mol with the larger membrane), i.e., the energies were estimated to be 30–40 % larger in the 12 nm × 12 nm membrane compared with the previously used 7 nm × 7 nm membrane. This may be due to the increased number of PIP2 lipids in the larger membrane . There were 11 PIP2 lipids in each leaflet of the 12 nm × 12 nm membrane in the present study versus 4 in the 7 nm × 7 nm membrane [2 was not present , the binding energies were estimated to be two–three-fold smaller [PTEN [We have previously simulated membrane binding of C2× 12 nm) . With thmembrane . This sumembrane . When PI smaller . Negativ smaller  and expeer [PTEN . The dif2 (70:29:1) plasma membrane mimic was estimated to have a Kd-value of 0.04 ± 0.01 µM, corresponding to a free energy of −42 ± 1 kJ/mol at 293 K [2 (80:15:5) membrane. Part of this discrepancy may be ascribed to the higher content of PIP2 in the simulations compared with the experiment (5 mol% compared with 1 mol%), albeit PS content is lower in the simulations (15 mol% compared with 29 mol%). Moreover, it has recently been shown that the Martini 2 forcefield is prone to overestimate protein–lipid affinity for amphipathic helices [The free energy values we calculated are somewhat overestimated when compared with experimental estimates ,31,32,33 helices , which m helices . When coThus, while comparison of the absolute free energy values between our calculations and experiments is difficult, we are able to compare trends between them.PTEN) contributes to membrane binding on a PC/PS membrane with a Kd of 84 ± 9 µM (ΔG = −23 ± 1 kJ/mol) compared with a Kd of 2.9 ± 0.3 µM (ΔG = −31 ± 1 kJ/mol) for the full-length PTEN [PTEN, which is qualitatively consistent with this large contribution of C2PTEN to the binding energy of PTEN, as determined experimentally. Moreover, in our simulations, PtasePTEN must have the C2PTEN domain present to achieve its productive binding conformation, which confirms its role as a lipid anchor for PTEN.It has been shown experimentally that the C2 domain of PTEN  [SHIP2 in our simulations; albeit, we cannot explain the observed decrease in binding energy upon addition of C2SHIP2, which was observed experimentally [SHIP2 domain, suggesting that the C2 domain does not act as a lipid anchor in SHIP2.In contrast to PTEN, the addition of the C2 domain to a Ptase construct of SHIP2 experimentally showed no increase in binding affinity to PIPThus, our results confirm that for PTEN, the C2 domain facilitates membrane binding and positioning the active site towards its substrate ,29,35. HSHIP2 is essential for cellular function [SHIP2 provides allosteric interdomain effects, which lead to an increase in the catalytic activity of the Ptase domain. With MD simulations, the authors demonstrated that an interaction between the C2 domain and R649 in the Ptase domain alter the dynamics of helices α5-7 (residues 615–643) and of loop L4 (residues 674–684). The authors proposed that the change in dynamics of L4 by C2SHIP2 is essential for catalytic activity by enabling L4 to alternate between an open and closed conformation [It has been shown by Le Coq et al. that C2function , and theormation . Here, wIn summary, our simulations support the putative role of C2 in SHIP2 as an allosteric regulator of the Ptase domain.Although SHIP2 and PTEN are both phosphatases and contain a catalytic Ptase domain and an adjacent C2 domain, the role of their C2 domains appears to be different. The C2 domain of PTEN participates significantly in membrane binding and is important for the positioning of the active site in the Ptase domain towards the lipid substrate—a role also observed for other C2 domains . In cont