Chloride ions play a pivotal role in scaffold assembly, based on studies of NC1 hexamers from mammalian tissues. First, Cl− activates a molecular switch within trimeric NC1 domains that initiates protomer oligomerization, forming an NC1 hexamer between adjoining protomers. Second, Cl− stabilizes the hexamer structure. Whether this Cl−-dependent mechanism is of fundamental importance in animal evolution is unknown. Here, we developed a simple in vitro method of SDS-PAGE to determine the role of solution Cl− in hexamer stability. Hexamers were characterized from 34 animal species across 15 major phyla, including the basal Cnidarian and Ctenophora phyla. We found that solution Cl− stabilized the quaternary hexamer structure across all phyla except Ctenophora, Ecdysozoa, and Rotifera. Further analysis of hexamers from peroxidasin knockout mice, a model for decreasing hexamer crosslinks, showed that solution Cl− also stabilized the hexamer surface conformation. The presence of sufficient chloride concentration in solution or “chloride pressure” dynamically maintains the native form of the hexamer. Collectively, our findings revealed that chloride pressure on the outside of cells is a primordial innovation that drives and maintains the quaternary and conformational structure of NC1 hexamers of collagen IV scaffolds.Collagen IV scaffold is a primordial innovation enabling the assembly of a fundamental architectural unit of epithelial tissues—a basement membrane attached to polarized cells. A family of six α-chains (α1 to α6) coassemble into three distinct protomers that form supramolecular scaffolds, noted as collagen IV E121–α556 , 14 as r− activates a molecular switch within NC1 domains that initiates oligomerization of two protomers forming an NC1 hexamer at the trimer–trimer interface (A)  (− ions belonging to groups 1 and 2 form a ring at the interface (A)   . In turntructure A  .Figure 1−-dependent mechanism is based on extensive studies of the chemical and physical properties of NC1 hexamers that were isolated from bovine and human tissues (B) . N. N−-depenterface , 19, 26.mb jelly  12). Th. Th−-dep− ions in assembly may shed light on functionality of collagen IV scaffolds in the genesis of tissues and organs and dysfunction in disease. Here, we developed a simple in vitro method of SDS-PAGE to determine the role of Cl− in hexamer stability. The methodology was amenable for a cohort of high-school and undergraduate students to participate in an expedition on Cl− function to the dawn of the animal kingdom. Collectively, their findings reflect a primordial function of chloride pressure that drives and maintains the assembly of collagen IV scaffolds.Knowledge of the evolutionary role of Cl− stabilizes hexamer quaternary structure in bovine and human tissues. We found that depletion of Cl− induced the dissociation of lens capsule BM (LBM) α121 hexamers  under nondenaturing conditions to ascertain whether Clhexamers  and recohexamers , devoid in vitro method to measure Cl− impact on hexamer stability using SDS-PAGE. Serendipitously, we found that the native hexamer is stable at room temperature in 1% SDS, a protein denaturant, and in the presence of 150 mM Cl−. However, in the absence of Cl−, the hexamer dissociates into subunits. These observations provided a strategy to modify the traditional SDS-PAGE into a novel method for analysis of Cl− impact. Hexamers were excised from tissues by collagenase digestion in the presence of 150 mM Cl− to preserve the native structure harboring the Cl− ions (B). Samples were prepared for electrophoresis with or without Cl− and at room temperature or boiled just before starting the electrophoresis in the standard fashion. Boiled samples served as a positive control for fully dissociated subunits. Accordingly, Cl− was included or excluded from the gel and the running buffer. To demonstrate utility of this method, we analyzed uncrosslinked hexamer from LBM (left panel). A comparison of gel patterns of samples prepared at room temperature, with and without Cl−, revealed that Cl− stabilized the quaternary hexamer structure at room temperature. This finding is analogous to our previous study using SEC analysis of LBM hexamer under nondenaturing conditions (right panel), which showed that Cl− stabilized the hexamer and prevented dissociation into dimer subunits. Collectively, the results demonstrated the capacity of the SDS-PAGE method to distinguish the impact of Cl− on hexamer stability in the context of crosslinked hexamers.Given that crosslinked hexamers predominate in animal tissues, we developed a simple Cl− ions B. SampleThis simple assay could be applicable in studies of other small-molecule–dependent proteins and complexes. To our knowledge, no proteins have been reported that demonstrate SDS resistance with the requirement of supplementing specific solutes. It was previously shown that SDS resistance is a common property of kinetically stable proteins with a bias toward a very high content β-sheet structure . In our in vitro method of SDS-PAGE provided a strategy for a cohort of 23 high-school and undergraduate students to participate on an expedition to determine whether Cl− plays a role in the maintenance of hexamer structure in diverse animals ranging from humans to Ctenophores. In support of their research activities, students were mentored in professional development, self-discovery, and wellness (The simple wellness . Of noteB), as described in the − supplement to maintain the native hexamer structure that harbors Cl− ions for stabilization (B).Unless otherwise noted, a small piece of tissue or small number of animals  was homogenized and digested with bacterial collagenase to excise the native NC1 hexamer B, as deslization B.Figure −-independent stable hexamers, possibly pointing to specific isoforms of collagen IV in certain tissues. Cartilaginous fish, dogfish, not only revealed the chloride-dependent mechanism but also demonstrated a significant amount of kidney-extracted hexamer to be Cl− independent  autoimmune disease  mouse model . The results reflect a substantial decrease in number of crosslinks per hexamer and a concomitant increase in uncrosslinked hexamers in all tissues (B). We probed the hexamers with chain-specific antibodies and found that crosslinks were also diminished in the α345 NC1 hexamer (B).Here, we explored the Clse model  to diminSDS-PAGE . CompariSDS-PAGE A. The re tissues B. We pro hexamer B.Figure − on conformation, we measured the binding of GP antibodies to α345 hexamers from kidneys of WT and PXDN-KD mice (A and B). The ELISA results showed that antibody binding, in the absence or the presence of Cl−, was identical for naturally crosslinked and crosslink-deficient hexamers. This result demonstrates that Cl− pressure, alone and independent of sulfilimine crosslinks, is sufficient to stabilize the hexamer surface conformation (C and D), revealing a role of Cl− in stabilizing both quaternary structure and conformation. This finding further highlights the importance of chloride saturation on the hexamer structure and reflects the conformational plasticity of the hexamer surface . Cell membrane is not only an enclosure for biological processes but also an active gradient maker for molecules including chloride ions. Chloride concentration inside the cell is low but high in the extracellular space. Unique properties of chloride ions to accept multiple hydrogen bonds enabled protein evolution and collagen IV scaffold assembly and maintenance. The location and dynamic nature of “chloride pressure” emerged as an elegant way to signal assembly and stabilize collagen IV scaffolds on the outside of cells. In turn, the scaffolds enable the assembly of a fundamental architectural unit of epithelial tissues—a BM attached to polarized cells that enabled the genesis of tissues and organs and animal evolution . Yet, thMus musculus) kidneys were collected from humanely euthanized laboratory mice, WT strain C57BL/6 ; the NC1 hexamer of human (Homo sapiens) kidney was from a previously purified frozen stock (Bos taurus) kidneys were purchased from a local butcher shop in Nashville; marmoset kidneys  were collected with the approval from the SA Pathology/Central Adelaide Health Network Animal Ethics Committee in 2014 under approval #68/11.Murine  liver and kidneys were obtained from a farmer’s market in Arizona; chorioallantoic membrane was collected from 15-day fertilized eggs purchased from a local farm in Nashville.Rooster (Chelydra serpentina), liver of caiman (Caiman crocodilus), and decapitated green iguana (Iguana iguana) were purchased from Exotic Meat Market, Inc. (https://www.exoticmeatmarkets.com).Frozen food-grade meat of snapping turtle (Ambystoma mexicanum) and African clawed frog (Xenopus laevis) were gifts from Drs Prayag Murawala and Louise A. Rollins-Smith, respectively.Kidneys from axolotl (Danio rerio) material was a gift from Dr James G. Patton; banded killifish (Fundulus heteroclitus) was the field collected near Bar Harbor.Zebrafish  was a gift from Dr Michelle D Bailey.Dogfish  was purchased from Gulf of Maine, Inc (gulfofme.com).Sea squirt (Asterias rubens) was purchased from Gulf of Maine, Inc (gulfofme.com).Starfish (Mytilus edulis), sea clam (Spisula solidissima), and market squid  were purchased from a local seafood market in Nashville; quick gloss snail (Zonitoides arboreus) was field collected in Nolensville.Mussel (Lumbriculus variegatus) were purchased from Worm Man's Worm and Crickets Farm (https://wormman.com); leeches  were purchased from Speedy Worm & Minnesota Muskie Farm (shop.speedyworm.com).California blackworms (Dugesia tigrina) was purchased from Ward's Science ; horseshoe crab flatworm (Bdelloura candida) was purchased from Gulf Specimen Marine Laboratories, Inc .Culture of brown planaria (Brachionus plicatilis) were purchased from Reed Mariculture, Inc (reefnutrition.com).Rotifers (C. elegans) were collected from laboratory stocks; the NC1 hexamer of the large roundworm of pigs (A. suum) was from a previously purified frozen stock (Roundworms (en stock .Drosophila melanogaster) whole animals were a gift from Andrea Page-McCaw (Vanderbilt University), and bulk material was purchased as a crude insoluble fly extract from Drosophila Genomics Resource Center ; adult culture of brine shrimp  was purchased from Northeast Brine Shrimp, LLC (www.livebrineshrimp.com); cicada (Magicicada spp) was field collected in Nashville; American lobster (Homarus americanus) was purchased from a local farm at Bar Harbor.Fruit fly (Mnemiopsis leidyi) was field collected for a previous study (M. leidyi) was from a previous study (Comb jellyfish (us study . The purus study .Nematostella vectensis) was from a laboratory stock; condylactis anemone (Condylactis gigantea) was purchased from a local aquarium store; moon jellyfish (Aurelia aurita) was from a laboratory stock.Starlet sea anemone  with 0.5 ml of TBS with Tween-20 four times. At the final step, insoluble material was resuspended in 0.250 ml of collagenase digestion buffer  containing 0.1% Tween-20. Homogenate was pelleted by centrifugation at 16,000n buffer  supplemeg for 30 min. The supernatant was discarded, the pellet was resuspended in fresh homogenization buffer using Polytron, centrifuged at 10,000g for 30 min, and this step was repeated two more times. A fresh buffer (20 ml) containing 10 mM Tris–HCl, pH 7.5, 1% deoxycholate (dissolved before adding NaCl!), and 150 mM was prepared at room temperature (20–25 °C), and the pellet was resuspended and incubated at room temperature for 1.5 to 2 h while mixing. The insoluble material was pelleted by centrifugation at 12,000g for 30 min at room temperature, and the supernatant was carefully removed. The thoroughly drained pellet was resuspended in 20 ml prechilled 25 mM Hepes, 150 mM NaCl, 25 mM 6-aminocaproic acid, 5 mM benzamidine–HCl, pH 7.5, and centrifuged at 12,000g at 4 °C for 30 min. The supernatant was discarded, and the previous step was repeated two more times. The final pellet was resuspended in 10 ml of the same buffer supplemented with 10 mM CaCl2 and 6 μg/ml collagenase and tumbled in a sealed container at 37 °C for 16 h. Finally, the suspension was centrifuged at 12,000g for 30 min, and the supernatant containing the NC1 domain released by collagenase digest was collected for further purification and analyses.This is a modified protocol . All bufA regular Laemmli system of Tris–glycine SDS-PAGE  was modiIn special cases, a Bis–Tris system was used , where tUpon completion of electrophoresis, regular and chloride-supplemented SDS gels were subjected to heating in the running buffer containing 0.1% SDS for 3 min to ensure complete denaturation of SDS-resistant species. The gels were then cooled to room temperature and subjected to a regular procedure of transfer to nitrocellulose membrane and Western blotting using NC1-specific JK2 antibody from Yoshikazu Sado, Shigei Medical Research Institute. JK2 monoclonal antibody was raised to the NC1 domain of human α3 chain but exhibited broad reactivity toward NC1 domains from different animal species . Mouse aM. musculus) kidney and partially purified NC1 hexamer from a kidney of common marmoset (C. jacchus) were run on the regular and “salty” gels, blotted, and detected using the JK2 antibody. Purified NC1 hexamers from kidneys of human (H. sapiens) and cow (B. taurus) were run on the regular and “salty” gels and Coomassie stained.Straight collagenase digest of murine  as well as a 15-day-old chorioallantoic membrane were used to extract the NC1 hexamer. After collagenase digest, the NC1 hexamer was purified and probed on the regular and chloride-supplemented gels. The specific NC1 bands were detected on Western blot with the JK2 antibody.Liver and kidney of rooster (C. serpentina), liver from caiman (Caiman crocodilus), and heart and kidney from iguana (Iguana iguana) were collagenase digested to release the NC1 domain from the insoluble matrix. The supernatant was analyzed on the gels, and the NC1 bands were detected by the JK2 antibody on the Western blot.Muscle tissue from turtle (A. mexicanum) and African clawed frog (X. laevis) were homogenized and subjected to collagenase digest. The solubilized material was analyzed by Western blot with JK2 antibody.Kidneys from axolotl (D. rerio) material was homogenized, collagenase digested, and solubilized material was partially purified by SEC before Western blot analysis; banded killifish (F. heteroclitus) organs were homogenized, subjected to collagenase digest, and solubilized material was analyzed by Western blot with JK2 antibody.Zebrafish (S. acanthias) were purified and analyzed on the Western blot using the JK2 primary antibody.NC1 hexamers from lens capsule, kidney, and intestine of dogfish  was homogenized and subjected to collagenase digest. The soluble fraction was run on the gels, and the NC1 bands were detected by using the JK2 antibody after blotting. For chloride-supplemented SDS-PAGE instead of a regular Tris–glycine gel, we used Bis–Tris gel, which allowed better separation of the hexamer band from the dimer.Whole animal of sea squirt (A. rubens) gonads were found to be a rich source of the NC1 hexamer. Collagenase digest of homogenized gonads was directly analyzed on the gels, and the NC1 bands were recognized by the JK2 antibody after blotting.Starfish (M. edulis) and sea clam (S. solidissima) whole tissues were run on the regular and “salty” gels, blotted, and detected using the JK2 antibody. Partially purified NC1 hexamers from lens capsules of market squid  and from whole animals of the quick gloss snail (Z. arboreus) were run on the regular and “salty” gels, blotted, and detected using the JK2 antibody.Collagenase digests of mussel (L. variegatus), and leeches  were run on the gels and detected on Western blots by using the JK2 antibody.Collagenase digests of homogenates of segmented worms, California blackworms (D. tigrina) and horseshoe crab flatworm (B. candida) were collagenase digested and partially purified over the SEC. The hexamer peaks were concentrated and analyzed on the gels. Brown planaria NC1 domain was recognized by the JK2 antibody after blotting, whereas horseshoe crab flatworm NC1 domain was visualized in the gel using the 2,2,2-trichloroethanol (TCE) approach  was subjected to collagenase digest and partial purification on the size-exclusion column. The hexamer fraction was run on the gels, and bands of NC1 domain were detected on Western blot using the JK2 antibody.Whole animal homogenate of rotifera  (NC1 hexamer from roundworm lization . The NC1A. suum)  was analD. melanogaster) was run on regular and “salty” gels and detected using the TCE in-gel visualization (M. spp), brine shrimp , and American lobster (H. americanus) were run on the regular and “salty” gels, blotted, and detected using the JK2 antibody.Purified NC1 hexamer of fruit fly (lization . CollageM. leidyi) was run on regular and “salty” gels, blotted, and detected using the JK2 antibody.Partially purified NC1 hexamer of comb jelly (A. aurita), condylactis anemone (Condylactis gigantea), and starlet sea anemone (N. vectensis) were run on regular and “salty” gels, blotted, and detected using the JK2 antibody.Partially purified NC1 hexamers of moon jellyfish , and density profiles of individual lanes were measured using the ImageJ , version 1.53a software . DensityBinding of affinity-purified α3-specific GP autoantibody (0.2 μg/ml) was tested using ELISA . Polystyhttps://office.microsoft.com/excel), Gimp , and Inkscape  software.Data processing and figures were done using the Excel (Microsoft, All data are contained within the article or This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Among participants with T2D, further analyses showed a pattern of higher HbA1c among those with lower growth mindsets and self-efficacy toward their general health or blood glucose level. These findings identify the belief-domains that may pose barriers to necessary self-care behaviors, informing future interventions to promote improved diabetes care and management.Growth mindsets and self-efficacy beliefs have been known to predict and promote resilience, challenge seeking, and improved outcomes in areas such as education and intelligence. However, little is known about the role of these two potentially influential beliefs in the context of type 2 diabetes (T2D), specifically in terms of whether and in which domains  these beliefs—or lack thereof—is prevalent among individuals with T2D. Given the lifelong challenges that individuals with diabetes often encounter with managing their disease, many may slip into a conceding negative belief that their diabetes is “too difficult to control” or simply “out of their hands,” inhibiting proactive self-management efforts. Results from our study ( Although evidence-based lifestyle changes and pharmacotherapy can help reduce mortality and the risk of diabetes-associated complications, the success of such interventions hinges largely on individuals’ incorporating self-management behaviors in their lives. Many of these behaviors, however, are difficult to sustain over the long-term and require individuals to navigate complex barriers throughout their lifetime2. Those who initially endeavor to modify their lifestyles may often revert to previous habits, especially after encountering setbacks such as increased inconvenience, conflict with personal priorities, and unsatisfactory blood glucose control. Should unfavorable health outcomes persist over time, individuals may slip into a conceding belief that their diabetes is “too difficult to control,” that they must be “genetically prone” so there is little they can do to change, or that their diabetes is simply “out of their hands.” Clearly, setbacks in self-management comprise only part of the various factors that may lead to failure in blood glucose control. But it appears that beliefs that one’s diabetes—or more specifically, blood glucose—cannot be easily improved, controlled, or managed may be quite pervasive, offering a plausible perspective of individuals’ difficulties in achieving sustained glycemic control through self-management. Obtaining an empirical understanding of this conjecture is pivotal in laying the groundwork for novel strategies to support behavioral change as has been demonstrated in other domains like education3.The prevalence of type 2 diabetes (T2D) has become a serious threat to global health, with many diagnosed individuals experiencing significant challenges in maintaining adequate glycemic control6. In particular, fixed versus growth mindsets and self-efficacy (SE) beliefs hold especial relevance in contexts fraught with challenges8. Individuals with a growth mindset (GM) believe that a specific attribute or condition is malleable, leading them to embrace adaptive beliefs and behaviors 9. At the other end of the spectrum, individuals may have a fixed mindset (FM), believing that there is little they can do to change or improve within a certain area of function, which is further reinforced by poor performance or outcomes10. Individuals may hold differing mindsets in different domains , with mindset valence varying across the fixed-growth spectrum12.Research in the fields of psychology and healthcare suggests that people’s health-related behaviors are often influenced by their beliefs and expectations14) and, increasingly, in health-related domains , there has been limited research pertaining to diabetes. To our best knowledge, only one correlational study involving 91 adolescents with type 1 diabetes has reported the role of GM in the context of diabetes: compared to adolescents with a FM, those with a GM had better glycemic outcomes after transitioning to adult care, with greatest improvements in HbA1c (the blood test used to assess glycemic control) among GM-adolescents who had the poorest glycemic control (HbA1c > 10%) before transition18. Taken together, extant theoretical and empirical research indicating that a GM can predict and promote more resilience, challenge seeking, and positive outcomes19 suggests that instilling a GM among individuals with T2D pertaining to the control of their disease may prove a crucial precursor to successful diabetes management. Thus, there is much potential for evidence-based research to establish whether and where mindsets matter in the context of T2D.Although mindsets have been well researched in the domains of education and intelligence  results8, while GM emphasizes the process of effort and willingness to learn through setbacks rather than necessarily arriving at a desired outcome. Compared to GM, health LOC and outcome expectancies have been relatively well studied in cohorts with diabetes and may have limited potential for generating novel contributions in our current context. Notably, research on these constructs has reported mixed findings. A meta-analysis of health LOC, for instance, found weak correlation between external LOC and HbA1c and no correlation between internal LOC and HbA1c26. This led the authors to recommend excluding the use of health LOC measures in the design of diabetes care protocols. In another study, outcome expectancies were found to be only moderately correlated with diabetes self-care compared to self-efficacy27, while yet another study found that dietary outcome expectancies failed to predict dietary self-care28.At this juncture, we recognize that GM and SE are not the only belief constructs pertinent to diabetes management and glycemic control. Health-related psychological constructs such as health locus of control (LOC) and outcome expectancies may seem to share some similarities with GM as their positive orientations may predict, or at least be associated, with health outcomes. Nonetheless, these constructs have clear conceptual distinctions. Health LOC refers to beliefs about whether one’s health outcomes are attributed to internal factors  or to external factors 28, there is substantial opportunity for examining beliefs that may hold greater resonance and applicability in the context of diabetes management. Importantly, investigating both GM and SE allows us to explore the role of two potentially influential beliefs in the T2D context in order to better ascertain the patient segment needing additional support, and where and how that support may be directed. Thus, we aim to (1) examine whether individuals with T2D would tend to have less GM and lower SE compared to individuals without T2D and (2) identify the domains in which differences in beliefs are observed. Assessing psychological constructs at more general, broader-level domains may be suited for predicting more general patterns of behavior or outcomes across multiple contexts29. On the other hand, measuring beliefs at a more specific level may be more optimal for predicting specific behaviors and outcomes within a specific context8. Given the advantages of both broader and specific measures, we examined beliefs in general life and general health domains, as well as in condition-specific areas—namely blood glucose, blood pressure, and cholesterol levels . Of these, blood glucose is often more difficult to successfully control given that medication adherence alone is typically insufficient and must be accompanied by sustained lifestyle modifications32.Given the presently limited research on GM and the prominence of SE as a recognized mechanism of diabetes outcomesWe also examine how GM and SE beliefs toward one’s blood glucose and general health may correspond with blood glucose (measured using HbA1c) among participants with T2D. Additionally, we conduct secondary analyses to explore whether the duration of having T2D may differentially affect beliefs depending on optimal versus non-optimal HbA1c levels. In addressing these goals, we aim to identify the belief-domain(s) that may pose barriers to necessary self-care behaviors with the vision of informing future interventions to promote better diabetes care and management.This cross-sectional study was conducted online in Singapore between April–November 2021. To avoid response biases where participants’ ratings of their general life beliefs may bias or cloud their responses on health-related beliefs, the survey was administered over two parts. Part A included questions on general life beliefs and participant characteristics. Part B, administered a week later, focused more specifically on general health beliefs and condition-specific beliefs.33, it is essential to ascertain whether the effects observed in this study are unique to T2D status or may be attributed to having a chronic condition in general. To investigate this possibility, we also recruited individuals with self-reported HT/HL—chronic diseases that are frequently present in both T2D and non-T2D populations—regardless of participants’ T2D status. This approach would allow us to examine whether mindsets and SE matter (a) only for individuals with T2D, (b) for those with T2D and HT/HL, and/or (c) those with just HT/HL.  Participants with severe or confounding conditions  were screened out at the start of the survey.Given that many individuals with T2D commonly have other comorbidities 34 ; other studies segment participants into different 10-year ranges of onset categories primarily based on convenience37. HL, on the other hand, is more common in individuals above the age of 4039). Second, we considered data collection constraints given that online research panels often skew towards younger participants. Setting a higher minimum age  would severely limit our already constrained participant pool, especially after applying our health-related exclusion criteria. Taking these factors into consideration, a minimum age threshold of 40 for all participant groups would balance comparability across diseased and non-diseased groups and data collection feasibility concerns.Furthermore, as age is a non-modifiable risk factor that may invariably influence the results of this study given the chronic conditions under investigation, we applied a minimum age of 40 to reduce self-selection bias based on age-associated health conditions. In determining an appropriate age cutoff, we first considered the typical onset age range of T2D, HT, and HL, and applied a minimum age that would reasonably serve as a “common denominator” across these chronic conditions. . All procedures were performed in accordance with the approved guidelines and regulations. Prospective participants were provided with the study information prior to the survey and informed about the use of their answers for analysis under anonymity. All participants provided their online informed consent before commencing the survey. Bot detection was implemented in the survey to facilitate identification and exclusion of potential non-human survey completions.40. Measuring beliefs in one’s overall ability or confidence to perform effectively across various general situations or tasks , this 8-item scale has been previously demonstrated to have high construct validity and reliability29 and possessed high internal reliability in this study (α = 0.92). Responses were scored on a 5-point Likert scale (5 = strongly agree), with higher scores indicating greater general life SE.General life SE was assessed using the widely used New General Self-Efficacy scale (NGSE)41. The PHCS has been previously demonstrated to have high construct validity and reliability30 and possessed high internal reliability in this study (α = 0.81). Responses were scored on a 5-point Likert scale (5 = strongly agree), with higher scores indicating greater health self-efficacy.SE towards one’s general health was measured using the 8-item Perceived Health Competence Scale 42. Adapted from the PHCS, the PDSMS is a valid measure of diabetes SE that holds high validity and consistency, and has been recommended for use with other chronic conditions by replacing the word “diabetes” with the specific condition under investigation 42. The PDSMS possessed good internal reliability in this study . Responses were scored using the same 5-point Likert scale, with higher scores indicating greater SE toward the indicated condition.SE toward blood glucose, blood pressure, and cholesterol levels were each assessed using the 8-item Perceived Diabetes Self-Management Scale (PDSMS)12. This well-established 3-item scale measures whether one’s personal attributes are believed to be fixed or malleable  and possessed high internal reliability in this study (α = 0.86). Responses were scored on a 6-point scale (6 = very strongly disagree), with higher scores indicating a stronger general life GM.General life GM was assessed using the domain-general measure of implicit theories43. Originally comprising statements pertaining to intelligence, this 4-item scale has been adapted to different domains by replacing “intelligence” with relevant keywords appropriate to the research context . Here, we used “health level” to reflect the degree to which one believes their health is changeable . This scale possessed high internal reliability in this study (α = 0.91). Responses were scored on a 6-point scale (6 = strongly disagree), with higher scores indicating a stronger GM regarding their general health.Mindset towards one’s general health was measured using an implicit theories of intelligence scale that has been well validated with test–retest reliabilityglucose level”). Each respective condition-specific mindset scale possessed high internal reliability . Responses were scored on the same 6-point scale, with higher scores indicating a stronger GM toward that condition.Mindsets toward blood glucose, blood pressure, and cholesterol levels were measured using the same scale assessing general health mindsets. As before, “intelligence” was replaced with condition-specific words to reflect the degree to which one believes that their blood glucose, blood pressure, or cholesterol is changeable  and weight (kg), from which we obtained their body mass index (BMI).Since we were interested in exploring the relationship between beliefs and blood glucose levels, we asked participants with T2D to indicate their most recent HbA1c reading. Given the possibility that duration of T2D status may impact health beliefs, we also asked participants to indicate the year in which they were diagnosed with T2D.p < 0.001). As such, we used quantile regressions to examine whether and in which domains would GM and SE beliefs differ between participants with T2D and those without T2D.Statistical analyses and data visualization were performed using SPSS version 27 and R 4.1.0. Based on preliminary linear regression analyses, we suspected a violation of the normality assumption which was confirmed by Kolmogorov–Smirnov tests on the linear regression residuals  but included HT/HL status in the model to accurately isolate the effect of T2D while accounting for HT/HL. We controlled for age, sex, ethnicity, marital status, education, employment, housing type, religion, and BMI in all analyses.We conducted quantile regression models for each belief-domain dependent variable . Each model initially comprised the following primary predictors: T2D status , HT/HL status , and their interaction (T2D × HT/HL). Including the interaction term allowed us to account for potential conditional effects . Subsequently, we dropped the interaction term if it was non-significant  and HbA1c among participants with T2D, we ran a nonparametric splines regression model for each belief–HbA1c relationship , adjusting for the same covariates as before. This model allowed us to identify the relationship structure and account for a possible non-linear structure.1. For T2D duration, we used a dichotomous indicator with a duration of ≤ 3 years as the cutoff for more recent diagnosis compared to those living with T2D for a longer time44.It is plausible that individuals who have been diagnosed with T2D for longer may tend to have more negative mindsets and SE beliefs towards their health and/or blood glucose. To explore whether T2D duration has an effect on health and/or blood glucose beliefs and whether the effect might differ for those with ideal versus non-ideal glycemic control we conducted a quantile regression to assess the two-way effect of self-reported HbA1c and T2D duration on all four combinations of belief-domain scores. Subsequently, we dropped the interaction term from each quantile regression model if it was non-significant. As before, we controlled for the same covariates. For glycemic levels, we used a dichotomous indicator with HbA1c ≤ 6.4% as the threshold for ideal glycemic control in diabetes care managementFrom the 1787 individuals who commenced the self-administered survey, a total of 903 participants completed the study (this sample attrition was due to the two-part study design as many dropped out before completing Part A or without participating in Part B). We excluded 10 participants who indicated other major/confounding conditions that were not automatically screened out at the start of the survey, leaving a total of 893 completed responses . We therefore excluded this interaction term in all quantile regression analyses reported in this study.The T2D × HT/HL interaction term was not significant for all our belief-domain DVs (ps ≥ 0.425). Instead, significant differences were observed in several health-related belief-domains. Compared to those without T2D, individuals with T2D were observed to have a significantly lower GM towards their blood glucose . Participants with T2D also had a significantly lower SE towards their general health , blood glucose , and cholesterol . There were no significant differences in GM towards general health, blood pressure, and cholesterol between individuals with T2D and those without T2D (ps ≥ 0.530). No significant differences were observed in SE beliefs toward blood pressure (p = 0.408). . Controlling for HbA1c without the interaction term, individuals with longer T2D duration  had a lower median GM towards their blood glucose compared to those with shorter T2D duration . T2D duration had no significant effect on general health GM, general health SE, and blood glucose SE (p ≥ 0.283).  general life encompasses a broad range of non-health related situations that extend beyond the immediate concerns of T2D management and (b) managing blood pressure may be perceived as less complex and demanding compared to blood glucose control , explaining why SE toward blood pressure did not differ between those with T2D and those without T2D.SE towards cholesterol, we should also observe significant associations between T2D status and GM towards cholesterol), the differences between GM and SE beliefs within specific health domains demonstrate nuances that distinguish these psychological constructs. For instance, beliefs that one’s cholesterol levels can be improved (GM) does not mean that one feels equally confident and equipped in performing the necessary actions for that change or improvement to happen (SE).While one might expect to observe consistent significant associations on the same domains across both GM and SE constructs . Moreover, blood glucose levels tend to worsen with increased duration of T2D, requiring ever more intensive pharmacological treatment and lifestyle modification. By implication, the challenge of managing blood glucose seems to rest substantially on the complexity of self-management behaviors of continued effort and readjustments, not just as an individual in functional daily living, but also within the interpersonal and social environment46. By extension, our findings may be generalizable to other chronic diseases  in which patients face recurring setbacks or that require self-care behaviors beyond medication adherence.Notably, given that blood glucose was the only domain consistently affected by T2D status across both GM and SE beliefs,individuals with T2D may struggle particularly with managing their blood glucose compared to managing their blood pressure or cholesterol levels. While high blood pressure and cholesterol may be more easily managed with medication adherence (with efficacy that is often sustained over time), medications to reduce blood glucose are less successful without lifestyle modificationsA low general health GM score of 2.30 had a corresponding HbA1c of 7.4%, while a higher GM score of 4.10 had a corresponding HbA1c of 6.5%.A low blood glucose GM score of 2.00 had a corresponding HbA1c of 7.2%, while the highest GM score of 6 had a corresponding HbA1c of 6.3%.A low general health SE score of 2.00 had a corresponding HbA1c of 7.7%, while a higher SE score of 3.50 had a corresponding HbA1c of 6.5%.A low blood glucose SE score of 1.00 had a corresponding HbA1c of 9.5%, while a higher SE score of 4.2 had a corresponding HbA1c of 6.7%.Although the effect sizes of T2D on the scores for the significant GM and SE health-related domains were not large, the scores were not homogenous across all individuals with T2D. Among participants with T2D, we observed a pattern of elevated HbA1c among those with lower general health and blood glucose GM or SE scores. Higher GM and SE scores were associated with lower HbA1c of a nadir of 6.3–6.7%. Namely:These observations suggest that patients with lower levels of GM and SE beliefs may benefit from improvements in these respective belief-domains . Naturally, our findings prompt next questions regarding causality: is high HbA1c the result of negative health-related beliefs, or do negative health-related beliefs arise from high HbA1c levels? While it is conceivable that a reciprocal relationship exists, where HbA1c levels and health-related beliefs influence and reinforce each other , we acknowledge that our study’s correlational design precludes claims about causality and prompts the need for prospective investigations to further ascertain the dynamics of these relationships.The exploratory results on T2D duration offer further nuance on individuals with T2D who might be more susceptible to fatalistic beliefs towards their blood glucose and suggest that lower GM beliefs may be “acquired” over time. Namely, those who have had T2D for more than 3 years tend to have less of a GM towards their blood glucose, regardless of their HbA1c control. These individuals comprise a sizeable segment who could potentially benefit from appropriate interventions on the malleability of their condition.48, it may be crucial to target beliefs that hinder self-management by equipping individuals with T2D to develop a greater GM and SE towards their blood glucose . Perhaps one way to break the vicious “negative beliefs ↔ poor glucose control” cycle is to address their mindsets toward diabetes and self-management efficacy. Doing so could potentially reduce psychological barriers toward necessary lifestyle modifications and medication adherence and thereby lead to improvements in HbA1c, reinforcing further self-efficacy and growth mindset beliefs in a positive cycle of change and diabetes control. Another approach is to employ short-term interventions that lead to visible decreases in blood glucose levels  to provide the needed physiological and psychological breakthroughs for individuals who believe that their blood glucose levels cannot be easily managed, disrupting their FM about the non-malleability of diabetes control.Because beliefs influence motivation and behavior in ways that can profoundly affect health outcomescondition, on affirming effort over outcomes, on how mistakes should be reframed, on how setbacks can serve as stepping-stones, on how feedback informs improvements, or their combination?). Fourth, although intervention effectiveness relies predominantly on patient participation, healthcare providers play a major role in influencing patients’ perspectives and intentions. For instance, making subtle changes in physician–patient conversations by framing information differently  can provide the psychological opportunities conducive for GM and SE beliefs to thrive49.Our findings emphasize several important areas for future efforts to explore how GM can be fostered among those with T2D. First, a crucial next step would be to explore and establish any causal relationship(s) between GM and T2D outcomes using experimental studies . Second, we need an improved understanding of the areas in which individuals with T2D feel resistant to change and action . Third, complementary research should identify key aspects or features of GM that would most effectively address those FM areas . Hence, we are unable to validate the actual conditions, beliefs, and HbA1c levels that they claim to have. We also do not have data on the dates of their latest HbA1c results and acknowledge that many non-critical healthcare visits would have been delayed or forgone during the Covid-19 pandemic. Hence, the reliability of these self-reported values should be approached with caution. Fourth, the study was limited to participants residing in Singapore, a country with a 14.9% diabetes prevalence in adults aged 20–79 and with one of the highest proportions of total deaths associated with diabetes (29%)Fifth, we also acknowledge that there are other psychological concepts that may be related to the concept of GM and SE—such as health LOC and outcome expectancies—that were not included in this paper. This may potentially limit our understanding of whether and to what extent these concepts intersect or interact with GM and SE, and how they might impact downstream health-related behaviors and outcomes.51. Importantly, knowing that such beliefs—consistently and particularly towards blood glucose—tend to be significantly lower amongst individuals with T2D provides important findings in explaining why managing diabetes may be particularly difficult for those with T2D. Evidence from this study offers a premise for future studies to not only probe the causality relationship between negative beliefs and blood glucose control, but also informs the direction and development of evidence-based behavioral change interventions.Taken together, this study breaks new ground in exploring whether and where GM and SE beliefs matter for people living with T2D, a condition that has become a growing global emergencySupplementary Information.


Systemic ATTR amyloidosis is an increasingly important protein misfolding disease that is provoked by the formation of amyloid fibrils from transthyretin protein. The pathological and clinical disease manifestations and the number of pathogenic mutational changes in transthyretin are highly diverse, raising the question whether the different mutations may lead to different fibril morphologies. Using cryo-electron microscopy, however, we show here that the fibril structure is remarkably similar in patients that are affected by different mutations. Our data suggest that the circumstances under which these fibrils are formed and deposited inside the body - and not only the fibril morphology - are crucial for defining the phenotypic variability in many patients. Hereditary ATTR amyloidosis has diverse disease manifestations. Using cryo-EM it was possible to reveal, that the phenotypic variations arise from the circumstances under which the amyloid fibrils are formed, rather than from different fibril morphologies. That is, different amyloid fibril morphologies have been found in patients or animals with different pathologies or clinical phenotypes. Examples hereof include the different tau fibril morphologies in different neurodegenerative diseases3, the different α-synuclein-derived fibrils in Parkinson’s disease and multiple system atrophy5, the different Aβ fibril structures in vascular and parenchymal amyloid deposits6, the different serum amyloid A protein-derived fibrils in the glomerular and vascular variants of systemic AA amyloidosis8 and the different prion protein fibrils in different strains of transmissible spongiform encephalopathies9.For several protein misfolding diseases it was found that the disease manifestations are correlated with the amyloid fibril morphologies that are deposited within the tissue10, which is natively a homo-tetrameric protein with a total mass of 55 kDa and 127 amino acid residues per protomer11. The general interest into this disease has greatly increased over the last decades, as systemic ATTR amyloidosis is aging-associated and as there are several disease-modifying pharmaceutical agents, which have become available to treat affected patients13.The involvement of clinically or pathologically variable disease manifestations is also characteristic for a hereditary form of amyloidosis caused by transthyretin amyloid protein (ATTR). ATTR amyloidosis is one of the major forms of systemic amyloidosis, raising the question whether its variability may also be provoked by different fibril morphologies. Systemic ATTR amyloidosis depends on the misfolding of transthyretin (TTR)15. There are early-onset and late-onset forms16, and it is thought that the patient’s phenotype is correlated to a certain allelic variant of TTR that is expressed in the respective patient17. Over 130 different mutational variants of the protein were found to be associated with systemic ATTR amyloidosis in addition to the wild type TTR (WT TTR)18. Many of these mutations were shown to destabilize the natively folded protein conformation such that they promote the dissociation and unfolding of tetramer20. But how these different mutations may lead to different disease manifestations and whether mutations and disease phenotype may be correlated with specific fibril morphologies is less well understood.The spectrum of disease manifestations observed in different patients affected by this disease includes polyneuropathy, cardiomyopathy, renal failure along with several other symptomsTo investigate the relationship between mutation, disease manifestation and fibril morphology, we have determined the cryo-electron microscopy (cryo-EM) structures of several ATTR amyloid fibrils, which were isolated from the amyloidotic tissue of patients with hereditary ATTR amyloidosis. The patients expressed different mutations of TTR protein and showed specific clinical presentations. Based on cryo-EM, however, we find essentially the same fibril structure in all patients, demonstrating that the mutations do not necessarily lead to different fibril morphologies.ATTR amyloid fibrils were isolated from the explanted hearts of three patients that were diagnosed with systemic ATTR amyloidosis. Each patient was heterozygous for a different mutational variant of the TTR protein . Therefore, all three patients expressed WT TTR along with a different mutational variant of this protein. The patients varied also in the exact clinical and pathological disease manifestations, although all patients showed severe cardiomyopathy. The first patient (TTR V20I) is a man who was diagnosed at the age of 56. He underwent cardiac surgery one year later, due to acute heart failure, but no additional symptoms were noticed. The second patient is a woman (TTR G47E) who was diagnosed at the age of 56 years. She suffered from carpal tunnel syndrome and severe cardiomyopathy and cardiac surgery was performed six years later. The third patient is a man (TTR V122I) who was diagnosed at the age of 51 years. He also showed carpal tunnel syndrome and received a heart transplantation three years after diagnosis. None of the patients received anti-amyloid medication until heart transplantation.21. This method avoids harsh denaturing conditions and allowed us to obtain large quantities of elongated amyloid fibrils. TEM analysis of negatively stained and cryo-frozen fibril samples showed an almost monomorphic fibril distribution  maps of the fibrils Fig.  and 2. F25 and found to range from 0.5 Å to 0.7 Å for the three structures. Each fibril protein contains thirteen β-strands that participate in the formation of thirteen cross-β sheets  contain fibrils that correspond to the one described here. We conclude that the respective mutations do not generally promote a different fibril structure and that patient-specific factors or circumstances are responsible for the formation of the altered fibril protein fold.Yet, it is possible that patients with mutant TTR proteins show structurally altered fibrils. For example, it was found that some patients who express TTRV30M and TTRY114C show fibrils that consist mainly full length TTR protein. These fibrils differ from the fibrils that occur in the vast majority of ATTR patients, including the three presently studied mutations - and also other patients expressing TTRV30M or TTRY114C26. This patient also showed conformational heterogeneity in a small segment of the ordered fibril core (residues R49-N68) that occurred immediately C-terminal to an internal disordered segment  and of Ulm University (103/21). Informed consent for publication of patient details was obtained from the patient, as well as consent for the use of these samples for scientific research.This study is based on analyses of cardiac tissue from explanted hearts obtained from three patients with hereditary ATTR amyloidosis. For each patient, the four exons of the transthyretin gene were sequenced from blood samples to identify the genetic variants. Samples from patients ATTRV20I and ATTRG47E were used in a previous study21. In brief, 125 mg frozen human heart tissue were diced with a scalpel and washed with 500 µL ice cold tris hydroxymethyl aminomethane (Tris)-calcium buffer . The sample was centrifuged for 5 min at 3100 × g and 4 °C. The supernatant was removed, and the pellet was resuspended in 500 µL ice-cold Tris-calcium buffer. The suspension was homogenized with a Kontes Pellet Pestle and centrifuged again for 5 min. This Tris-calcium buffer washing step was repeated four more times. The pellet of the last centrifuge step was resuspended in 1 mL freshly prepared 5 mg/mL Clostridium histolyticum collagenase (Sigma) in Tris-calcium buffer with ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor (Roche). The solution was incubated overnight on a shaker at 37 °C and centrifuged for 30 min at 3100 × g and 4 °C. The supernatant was removed, and the pellet was resuspended in 500 µL Tris-EDTA buffer  and centrifuged for 5 min at 3100 × g and 4 °C. This Tris-EDTA buffer washing step was repeated two more times. The pellet was finally resuspended in 100 µL ice-cold water and centrifuged for 5 min at 3100 × g at 4 °C. The fibril-containing supernatant was retained. The water extraction step was repeated five more times to generate six supernatant fractions.Amyloid fibrils were extracted from the collected tissue, according to a previously described extraction protocol6 ions with a maximum fill time of 500 ms. The raw data was deconvoluted by the MASH Explorer41 using default settings and the “Quick Deconvolution” feature. To avoid artefacts, the deconvoluted spectra contain only peaks, where the monoisotopic masses could be assigned with confidence score of at least 90% and which resulted from m/z peaks with 5 charge or more. Masses were manually assigned to the WT and variant TTR sequences using the software mMass v5.5.042.A 500 µL aliquot of each fibril solution was lyophilized and resuspended in 6 M guanidine hydrochloride, buffered with 50 mM Tris-HCl and pH 8 to reach a protein concentration of 0.5 mg/ml. After an over-night incubation step, an aliquot containing 1 µg of protein was adjusted to 15 µL using 0.1% (v/v) trifluoroacetic acid (TFA), and was applied onto a U3000 RSLCnano (Thermo Fisher Scientific) column. The eluate was applied online onto an LTQ Orbitrap Elite system (Thermo Fisher Scientific) that was equipped with a nano-electrospray ion source and distal coated SilicaTips . The samples were analyzed in the positive ion mode using a spray voltage of 1.5 kV. The mass spectra were acquired from 370 to 1700 m/z in the ion trap of the instrument using a normal scan mode. The resolution was set to 30,000 (at 400 m/z). The automatic gain control was enabled and set to 10To prepare the analysis specimens formvar/Carbon 200 mesh copper grids (Electron Microscopy Sciences) were glow-discharged at 20 mA for 40 s with a PELCO easiGlow instrument (TED PELLA). A 3.5 µL aliquot of the fibril solution was placed onto the grid. After incubation of the sample for 45 s, excess solvent was blotted away with filter paper (Whatman). The grids were stained three times with 10 μL of a 2% (w/v) uranyl acetate solution. The grids were imaged with a JEM-1400 TEM (Joel) that was operated at 120 kV and equipped with a F216 camera (TVIPS).Formvar/Carbon 200 mesh copper grids (Electron Microscopy Sciences) were glow-discharged for 40 s using a PELCO easiGlow™ glow discharge system operated at 20 mA. A 3.5 µL aliquot of the fibril sample was applied onto the grid and excess solvent was blotted away with filter paper. The grids were dried at room temperature. Using a Blazers TKR 010 instrument, a 1 nm thick layer of platinum was evaporated at an angle of 30°. The grids were analyzed using a Hitachi S-5200 scanning electron microscope (Hitachi) at 10 kV acceleration voltage.43.C-flat 1.2/1.3 400 mesh holey carbon cupper grids (Electron Microscopy Sciences) were glow-discharged for 40 s using a PELCO easiGlow™ glow discharge system operated at 20 mA. The grids were mounted in an EM GP2 (Leica) and a 3.5 µL aliquot of the fibril solution was applied before they were blotted with filter paper for 4 s at 21 °C and 90% relative humidity. The grids were plunge frozen in liquid ethane and controlled, if necessary, with a 2100 F transmission electron microscope (Joel). Details of the cryo-EM data collection is listed in Supplementary Table 44 and in case of the falcon 4 data, the movie frames were summed into a single micrograph using the RELION 3.1 EER implementation45. Motion correction and dose-weighting was done using MOTIONCOR 2.146. The contrast transfer function was estimated from the motion-corrected images using CTFFIND-4.147. All subsequent image-processing steps were performed using the helical reconstruction methods implemented in RELION 3.149. The fibrils were picked manually and extracted with a box size of 256 pixel and an inter box distance of 10%. A reference free 2D classification was performed to remove low-quality segments. The remaining segments were subjected to a 3D classification using a featureless cylinder as the starting reference structure. A combination of 3D classification and 3D auto refinement steps were carried out to select the best segments. contrast transfer function -refinement50 and Bayesian polishing51 were performed to further increase the resolution. See Supplementary Table The raw data move frames were gain-corrected using IMOD25. Each model was built individually. The manual refinement was done using Coot52 with non-crystallographic symmetry constraints, Ramachandran, atomic displacement parameter and rotamer restraints. The atomic clashes, rotamer and Ramachandran outliers and model geometry were analyzed by the validation output generated using MolProbity53 and the comprehensive validation tool in Phenix54. Once a satisfactory main and side-chain density fit was achieved for one polypeptide chain, a fibril stack comprising six poly-peptide chains was assembled using the pdbsymm tool implemented in Situs55. The described cycle of iterative refinement and modeling was repeated for the fibril stack until a reasonable density to model fit was achieved. Supplementary Table The previously described structure of the ATTRwt amyloid fibril, protein data bank (PDB) entry 8ADE, was used as a starting structure and altered, if necessary, at the position of mutation using ChimeraFurther information on research design is available in the Supplementary InformationPeer review fileReporting SummarySource Data


In the case of tomato, increases in yield by as much as 80% are observed when plants are cultivated at 1000 ppm [CO2], which is consistent with current commercial greenhouse production methods in the tomato fruit industry. These results provide a clear demonstration of the potential for elevating [CO2] for improving yield and quality in greenhouse crops. The major focus of this review is to bring together 50 years of observations evaluating the impact of elevated [CO2] on fruit yield and fruit nutritional quality. In the final section, we consider the need to engineer improvements to photosynthesis and nitrogen assimilation to allow plants to take greater advantage of elevated CO2 growth conditions.Several long-term studies have provided strong support demonstrating that growing crops under elevated [CO Early work in the 1980’s suggested that e[CO2] increased the average yield of all plants tested by approximately 30%, with optional [CO2] concentration for growth and yield in the range of 700 to 900 ppm with concentration in excess of 1000 ppm having a negative impact on plant growth and yield [2] (800–900 ppm) increased lettuce, carrot, and parsley yield by 18%, 19%, and 17%, respectively in greenhouse grown crops. However, the yields of leek, chinese cabbage and celery were not significantly affected by increases in growth [CO2] concentration [e[CO2] results in an increase in vegetable number (yield) by on average 32% and vegetable mass by 11% [e[CO2] has both positive and negative impacts on vegetable quality. For example, whilst concentrations of fructose (+14.2%), glucose (+13.2%), total soluble sugar (+17.5%), total antioxidant capacity (+59.0%), total phenols (+8.9%), total flavonoids (45.5%), vitamin C (+9.5%), and calcium (+8.2%) increased in the edible part of vegetables, protein  nitrate (−18.0%), magnesium (−9.2%), iron (−16.0%), and zinc (−9.4%) decreased [e[CO2] , legumes, and maize-have a 4–10% reduction in iron concentrations of when grown under e[CO2] (~550 ppm) [e[CO2] can positively and negatively impact on legumes, grain and vegetables on a crop-by-crop basis and simultaneously alter quality attributes in the same harvestable material.The majority of research evaluating the impact of nd yield . In the 550 ppm) . These r2] on fruiting crops production in commercial growing systems. This paper examines these studies and the long-term implications of e[CO2] on the yield and quality of fruit required to feed a growing population. In the last section, we discuss the potential for designing crops for these new growing environments and allowing them to take full advantage of the introduced CO2, potentially increasing crop yield, reducing costs for commercial producers, and improving quality of the final product providing high nutritional value to consumers.The aim of this review is to provide an overview of the current available data of the impact of elevated [COe[CO2], in some cases as high as 2000 ppm. The effects of e[CO2] of fruit yield and quality has been extensively studied  has been reported [e[CO2] were found to be preferable for consumption in sensory panels [2] could contribute to an increase in Vitamin C improving their nutritional value for the consumer. However, growth at e[CO2] does not have the same impact on all species, as another studies in barley reported a significant decrease in Vitamin C content [e[CO2] and suggesting that high carbon growth environments may not always provide the best outcome for the consumer even though increases in yield maybe the producers primary concern  when the concentration was increased to 700 ppm, suggesting that the level of CO2 enrichment impacts fruit quality and careful consideration is needed to ensure an appropriate balance between levels of e[CO2] and final yield. Lycopene is an important phytonutrient, is sold commercially as a dietary supplement, and has been reported to possess anti-cancer properties and can improve cardiovascular health  of 800-900 ppm, in addition to a 28% increase in vitamin C at ripe stage and an ~8% increase in total soluble solids . These yield increases were in part attributed to an increase in the size of fruits [e[CO2] (750 ppm) [e[CO2].  , indicat750 ppm) , 52. Thi750 ppm) , reducin[e[CO2]. .e[CO2] can have both a positive influence on yield and nutritional quality, however, growth at [CO2] levels above what is optimum can negatively impact some quality traits.These reports suggest that the effects of growing crops in Rosaceous crop research in this area has focused primarily on cultivated strawberry with a small number of studies on raspberry and Nashi pear . This ise[CO2] of 450 ppm – 3000 ppm, while dry fruit yield increased by up to 120% , fruit yield increases for plants grown in enriched-carbon atmospheres ([CO2] = 450 ppm – 3000 ppm) ranged between 16.2% and 41% in the absence of other parameters that could alter fruit yield. In high nitrogen supplemented fertilisation, fruit yield was as high as 106% when grown under e[CO2] of 800 ppm [2] enrichment to unlock the greatest yield increases in cucumber. Interestingly, when grown under e[CO2] of 1200 ppm with the addition of high nitrogen fertilisation treatment, studies found a yield increase between 71% – 73% [e[CO2] of 800 ppm. Concentrations of [CO2] above optimal reduced stomatal density, stomatal conductance (gs), the maximum carboxylation rate (Vcmax) and the maximum photosynthetic electron transport rate (Jmax) [e[CO2], however only phosphorus showed a significant reduction in multiple cycles [e[CO2] may enhance fruit flavour and fruit yield at the expense of nutritional value.In cucumber  . This sue (Jmax) . The inoPrunus avium L.) plants were grown for 19 months in climate-controlled greenhouses at ambient  or e[CO2] (700 ppm). Elevated [CO2] treatment increased photosynthesis and dry matter production, leaf (55%) and stem (61%), after two months at 700 ppm, however, this initial stimulation is not sustained. Photosynthetic rates were less after 10 months of growth than after 2 months of growth, and only small increases in dry mass are still evident after 10-months, suggesting that sweet cherry acclimates to e[CO2] due to long-term exposure [e[CO2] on fruit yield or quality. In Nashi pear, a CO2-enriched atmosphere of 700 ppm increased fruit weight, diameter and length along with a 22.5% increase in Brix, . Howeve2-enriched growth for improving photosynthesis, increasing yield and quality of tree crops. However, they also suggest that some crops, especially perennial crops, may become acclimated to higher [CO2] and any gains may be lost over time.While these studies are limited in, they do indicate the potential of CO2 uptake could have other benefits. It is notable that growth of fruit crops in carbon enriched atmospheres has a similar effect of protecting against environmental stresses, such as drought and elevated temperature, that may become increasingly common due to climate change as plants genetically engineered to increase carbon assimilation. For example, in melon (Cucumis melo), growing plants in e[CO2] has been shown to mitigate yield losses from increased salinity [e[CO2] of 800 ppm was sufficient to rescue any significant yield loss of total and marketable fruits from salinity stress (20 mmol L−1 NaCl)  was insufficient to protect yields from drought conditions triggered by higher temperatures demonstrating that benefits in some crops may not be translatable across all crops of agronomical importance. Furthermore, in tomato plant Zhou et al. [e[CO2] were more sensitive to combined drought and heat stress; e[CO2] drives gs and transpiration reducing net photosynthesis and therefore productivity, which is concerning given that greenhouses tend to have elevated temperatures compared to the external environment due to the nature of their construction, glass and metal, and therefore e[CO2] in an enclosed system may negatively impact on yields if water supplies are limiting. This demonstrates that irrigation within greenhouse environments is an essential element and adjusting water regimes to maintain productivity and optimise water-use efficiency.The work presented above also suggest that increasing COsalinity , and in −1 NaCl) . It couly et al.  demonstru et al.  showed t2] that causes the increase in air temperature  by absorbing energy and preventing it from being radiated out into space ; as; as2] th2 assimilation rates by increasing the regeneration of the Rubisco substrate RuBP [2] for photosynthesis or increasing the plants’ ability to assimilate [CO2] under ambient conditions could have a similar protective effect. It would be interesting to explore if increased carbon assimilation rates, through atmospheric manipulation or genetic modification, can have a positive impact on crop resistance to high salt environments and other abiotic stresses in large field trials or commercial greenhouses. There is currently evidence that over-expressing the Calvin-Benson cycle (CBC) enzyme SBPase can increase tolerance to chilling stress in tomato [2 uptake could mitigate against the effects of global increases in temperature under e[CO2]. This may be deemed especially important given the expected impact of global climate change. This suggests that increasing carbon assimilation through manipulation of photosynthesis  surpasses 550 ppm, satA will be limited by the rate of RuBP regeneration rather than Rubisco activity suggesting there is scope to improve plant photosynthesis to increase yield in greenhouse environments where CO2 is routinely increased to 1000 ppm or more for short periods of time. These short time-periods are furthermore unpredictable and chaotic given that greenhouses must be vented, due to external environmental conditions, to maintain, as close as possible, optimal growing conditions i.e temperature and humidity inside the growth facility. Furthermore, the [CO2] dosing capacity must be economically beneficial, especially given the chaotic nature of CO2 loss to the environment during periods of venting. As dosing increases, costs go up accordingly determined by the cost of CO2. Moreover, at some point, there is a price limit where the supplemental cost of CO2 increases to a point where costs cannot be recovered by the selling price of the product. In the last year, CO2 costs have increased for £100 per tonne to as much as £3000 per tonne [e[CO2], or maintaining higher yields when CO2 costs are unmanageable become more important.As  enriched environments such as greenhouses are discussed below . Plants with higher NUE may allocate N toward both the photosynthetic complexes  and/ore[CO2]. A recent report of a large grain rice cultivar, Akita 63, having a high yield due to an enlarged sink capacity without and photosynthesis improvement. However, this work demonstrated that source capacity was strongly limiting the yield potential under high N fertilization. These authors suggested that enhancing photosynthesis is an important step to further increase yield of current high-yielding cultivars [e[CO2] could provide a step-change in yields in greenhouse cultivated crops.A recent review has identified a number of targets in the literature to improve N uptake, assimilation and remobilisation through genetic manipulation  throughout the vegetative phase ) [2 assimilation in supplemented [CO2] growth environments.One potential target for genetic manipulation is the starch synthesis enzyme adenosine diphosphate glucose pyrophosphorylase (AGPase); increasing AGPase activity has potential to increase starch accumulation for growth. Increased accumulation of starch has been shown to have little negative feedback on photosynthesis  and incr2]) , suggest2] environments, allowing them to take better advantage of supplemental CO2, increasing net photosynthetic rates and associated yields  [e[CO2], it seems plausible that cucumber fruit photosynthesis may contribute directly to fruit size (and therefore yield by weight) and quality through their ability to directly access carbon in an enriched atmosphere via their stomata  , 134. Whsis, see , 135. Thsis, see ) to bala2] concentrations on a crop-by-crop basis, to maximise productivity. An evaluation of fruit quality under these conditions has also been shown to be highly variable between treatments and difference are observed between cultivars with the same treatment suggesting that much more research is required to identify the specific mechanisms behind changes in fruit quality. In the case of soft fruit production in greenhouse environments, it will be important to determine if the quality of fruit harvested early in the season differs from that of fruit harvested later in the season when plants have spent a more significant period of time exposed to e[CO2] growth conditions. Cherry for instance, when grown under prolonged periods of e[CO2], acclimates to prolonged exposure and initial significant gains in yield observed after two months are less detectable after ten months and are not significantly different to control plants grown at a[CO2]  . This mae[CO2] controlled environments may be about more than additional carbon. Controlled environments also allow the regulation of transpiration (e.g. by controlling vapour pressure deficit) and therefore water uptake and the inclusion of additional fertilisation . Breeding new varieties adapted to these growth conditions may also be more amenable given the hostility towards genetically modified crops. A recent review noted that new phenomics, genomics, and bioinformatics tools make it possible to harness the untapped potential of crop genetic resources (including wild relatives) to create combinations of traits to enhance yield in high [CO2] controlled environments [Increases in yield associated with ronments .2] growing environments traditionally used in greenhouses. However, over the last several decades, agricultural research has adopted technologies such as genetic engineering and “genome editing” to improve traits in key crops that could be useful in these circumstances [Breeding alone may not be sufficient to adapt all varieties, or all crops, to high [COmstances , 138–140mstances  and tissmstances . If the Genetic manipulation, may need to go beyond the direct manipulation of carbon assimilation in leaves , 180, buIt should also be noted that the introduction of new growing, hydroponics, aquaponics and aeroponics may require further study, to breed and adapt or engineer plants root architecture for these new growth media. In conclusion, greenhouse cultivation offers the opportunity to manipulate growing atmosphere, lights and VPD for improved yields and we can now look at the opportunities to breed and engineer plants specifically optimised for these conditions.This research is funded by the Biotechnology and Biological Sciences Research Council (BBSRC) Collaborative Training Partnerships (CTP) for Fruit Crop Research in partnership with NIAB EMR and Reading University. N.H.D was supported by “Realising increased photosynthetic efficiency to increase strawberry yields”  awarded to A.J.S. A.J.S is supported by the Growing Kent and Medway Program, UK; Ref 107139. N.H.D and A.J.S drafted and wrote the manuscript with input from T.L, C.A.R and C.W who also edited the final version.The authors declare no competing interests.


Nevertheless, information on radon levels of natural waters and the potential human exposure risks associated with their domestic use is very sparse. To elucidate the environmental factors increasing human exposure risk to radon during domestic water use, we undertook a survey of local water sources, including springs, rivers, wells, and boreholes, over different temporal scales. Continental waters were highly enriched in 222Rn: activities ranged from 1.2 to 20.2 Bq L−1 in rivers and levels one to two orders of magnitude higher were found in groundwaters . The geology and hydrogeology of local crystalline aquifers support one order of magnitude higher 222Rn activities in groundwater stored in deeper fractured rock compared to that contained within the highly weathered regolith at the surface. During the mean dry season, 222Rn activities nearly doubled in most sampled waters in comparison to the wet period . Seasonal water use and recharge cycles and thermal convection are postulated to explain this variation in radon activities. The high 222Rn activities cause the total effective dose of radiation received from domestic use of untreated groundwaters to exceed the recommended 0.1 mSv y−1. Since more than 70% of this dose comes from indoor water degassing and subsequent 222Rn inhalation, preventative health policy in the form of 222Rn remediation and mitigation measures should be implemented prior to pumping untreated groundwater into dwellings, particularly during the dry period.The Ría de Vigo catchment is situated in the largest radon-prone area of the Iberian Peninsula. High local indoor radon (The online version contains supplementary material available at 10.1007/s11356-023-27305-6. The short half-life of 222Rn (3.8 days) means that it is a continuous source of natural radiation. The decay of radon emits an α particle following a chain that also includes the short-lived, α-emitting 218Po, 214Po, and 210Po . Although 222Rn found in air or water originates primarily from the local geology, both environmental levels and exposure routes differ according to the medium, and consequently, mitigation actions are distinct. Despite epidemiological and physiological evidence of the risks associated with human exposure to 222Rn derived from domestic water use , semi enclosed embayment in the northwestern coast of the Iberian Peninsula  that usually reach the soil surface Fig. . QuaternThe local aquifers are configured in a two-layered system based on the dominant weathering zonation: the upper aquifer is contained within the highly weathered regolith that presents variable thickness typically from 5 to 20 m depth Fig. . These un = 2). Thus, additional spring samples (n = 5) were collected during the winter/spring period (May) in 2021 , while the second survey was before the fall rain season began . Water samples were collected from 8 rivers, 7 springs, 16 wells, and 14 boreholes throughout the basin  was used to determine 226Ra activity during the summer/autumn basin-wide (dry season) survey. Two treatments were used on replicate samples; both groups were first purged with 222Rn-free air and one of them was subsequently filtered through GF/F filters  to remove particles. Filtered and unfiltered samples were then stored in 250-mL gas-tight bottles for at least 30 days  to allow secular equilibration between 222Rn and 226Ra. The 226Ra activity was then determined based on the ingrowth of 222Rn activity during storage.Additional water samples were collected for the determination of 222Rn-in-air levels, a private bathroom (1.4 × 2.6 m), equipped with a shower using the semi-public water supply system monitored on a weekly basis in 2019 was repurposed into a large degassing chamber. Water was allowed to flow through the shower head for different periods of time  at a constant water flux (7.3 L min−1) while air inside the bathroom was pumped through the sampling loop of a RAD7 radon monitor recording for 5-min cycles during a total of 85 min. Radon background levels inside the bathroom were measured for 10 min prior to the shower being turned on. Temperature of water flowing through the shower was 16.0 °C.To illustrate the effect of residential groundwater use on domestic https://www.meteogalicia.gal). The discharge of the basins’ main rivers was obtained from the public gauging station network managed by Augas de Galicia .A high-resolution terrain model of the study area was used to obtain the altitude of the sampling points and the river catchment areas. Climatological data  during the period covered by this study (2018–2021) was obtained from the public network of meteorological stations (13 stations in the study area) managed by MeteoGalicia  regular, when the water supply is used in the household in a volumetrically sustained way throughout the year; (b) irrigation, when groundwater supply is used for both household activities and irrigation, i.e., water consumption increases during the dry months; and (c) sporadic, when groundwater is not regularly used in the household.222Rn and its short-lived decay products derived from domestic water use was calculated according to the United Nations Scientific Committee on the Effects of Atomic Radiation report , −1 and −1 m−3)−1), 222Rn indoors (10−4), F is the equilibrium factor between 222Rn and its progeny (0.4) and O is the typical indoor occupancy (7000 h y−1). 222Rn during the activities that use water  and −1) taken from the ICRP , comparable to those measured in springs . The highest mean 222Rn activities are observed in groundwater samples collected from boreholes screened in the fractured rock aquifer layer .The radon levels in rivers of the Ría de Vigo basin range from 1.2 to 20.2 Bq Ln = 36; Wilcoxon signed rank test p < 0.001; average change: 2.8 ± 0.7 times; Fig. 222Rn activities from winter/spring to summer/autumn. The highest averaged seasonal changes are found in the sampled wells (4.0 ± 1.6 times), followed by rivers (2.7 ± 0.9 times), boreholes (1.9 ± 0.2 times), and springs (1.5 ± 0.1 times).Radon activities in continental waters of the Ría de Vigo basin were seasonally variable, with summer/autumn levels more than double those measured during winter/spring , groundwater samples frequently exceeded this threshold. During winter/spring, > 40% of wells and springs and 78% of the boreholes that we sampled had 222Rn in water activities above 100 Bq L−1. During summer/autumn, this incidence increased to 65% of the wells and springs (n = 17) and all boreholes (n = 11) that we sampled.While none of the surface waters we sampled reached the 100 Bq L226Ra in water measured during the summer survey were very low compared to 222Rn  for our dataset  with depth of well and borehole screening and altitude, while PCA component 2 is governed by location (latitude and longitude) and meteorological variables  and summer/autumn  over groundwater 222Rn levels has been extensively documented , larger contact of water with the atmosphere would entail faster groundwater 222Rn degassing compared to boreholes. During the dry months, higher water demand would imply shorter water residence times inside the wells and in contact with the atmosphere and could partially explain the observed increase in 222Rn activities measured during the summer  compared to those drawn from the fractured bedrock (boreholes). The faster recharge of the aquifer within the regolith compared to the fractured rock underneath . Within borehole MBH , and the stability of 222Rn activities, aside from autumn, suggests that groundwater dynamics  are of low magnitude there, following the vertical zonation of these properties in crystalline aquifers. There, the relative contribution of the two springs also used in the sampled water supply unit is expected to suddenly increase during rainfall events and would explain the sudden drop in 222Rn activities during autumn.Strong fluctuations in the measured 019 Fig. . A partiust Fig. . Beyond MBH Fig. , 222Rn a222Rn activity with respect to groundwaters characteristic of the study area  may explain the prevailing diluting effect of rainfall recharge on groundwater 222Rn levels observed in the Ría de Vigo basin.Groundwater degassing , a threshold that is seasonally surpassed in the study area. This has strong implications for human exposure to 222Rn from groundwaters due to the large vertical gradient in 222Rn content observed in the study area; groundwaters from the regolith can, in fact, contain much larger 222Rn activities than those supported by bedrock emanation, transporting 222Rn-rich waters from depth. Together with the current predictions of air temperature rise, a decrease in annual recharge, and a concentration of rainfall during winter and spring due to climate change, thus prolonging the duration of the dry season , 3 out of 4 sampled springs, and all the sampled boreholes (n = 14) would result in 222Rn ingestion radiation exposure exceeding the WHO’s recommended dose limit (0.1 mSv y−1). Furthermore, when considering the total radiation dose estimated from both ingestion and inhalation of 222Rn derived from domestic water usage, the WHO’s safety threshold for individual radiation exposure would only be cleared if river water were the sole source of domestic water supply. All the groundwater samples  represent sources of domestic water that would result in total effective radiation doses associated with 222Rn higher than 0.1 mSv y−1, up to a maximum of 5.7 mSv y−1  has set a maximum recommended radiation exposure of 0.1 mSv y222Rn in water used for domestic purposes come from indoor water degassing due to daily activities such as doing the laundry or showering. The activity of 222Rn in groundwater sourced from the borehole supplying the shower used to measure 222Rn levels inside the bathroom was 592 ± 11 Bq L−1 during the experiments. Once the shower was turned on, the 222Rn activities inside the bathroom rapidly increased . These data show that a large proportion of the 222Rn dissolved in water outgassed during shower use . Since the water used in our shower experiments was not heated, these degassing rates can be considered minimum values, as they are expected to increase with increasing water temperature Supplementary file2 (XLSX 21 KB)Below is the link to the electronic supplementary material.