Capparis spinosa is a widespread charismatic plant, in which the nocturnal floral habit contrasts with the high visitation by diurnal bees and the pronounced scarcity of hawkmoths. To resolve this discrepancy and elucidate floral evolution of C. spinosa, we analyzed the intrafloral patterns of visual and olfactory cues in relation to the known sensory biases of the different visitor guilds .C. spinosa. Additionally, we sampled visitation rates using a motion‐activated camera.We measured the intrafloral variation of scent, reflectance spectra, and colorimetric properties according to three guilds of known visitors of C. spinosa contained a typical sphingophilous scent with high emission rates of certain monoterpenes and amino‐acid derived compounds. Visual cues included a special case of multisensory nectar guide and color patterns conspicuous to the visual systems of both hawkmoths and bees.Carpenter bees visited the flowers eight times more frequently than nocturnal hawkmoths, at dusk and in the following morning. Yet, the floral headspace of C. spinosa. Our study revealed two interesting paradoxes: (a) the flowers phenotypically biased towards the more inconsistent pollinator; and (b) floral display demands an abundance of resources that seems maladaptive in the habitats of C. spinosa. The transition to a binary pollination system accommodating large bees has not required phenotypic changes, owing to specific eco‐physiological adaptations, unrelated to pollination, which make this plant an unusual case in pollination ecology.The intrafloral patterns of sensory stimuli suggest that hawkmoths have exerted strong historical selection on  Pollinators are agents of selection on floral phenotypes both at microevolutionary and macroevolutionary levels  with numerous protruding long stamens and styles, lacking a prominent nectar tube Werth, . AlthougCapparis spinosa L. (caper bush) is the most prominent member of the family, a plant with high phytochemical potential, economic value, and ethnobotanical significance dating to the Neolithic Period , in Koudouroudia , and in Mytilene ; the minimum distance between sites was 8.6 km, and the maximal was 20.7 km. Along this distance, the plants’ habit, landscape, elevation, climate (thermo‐Mediterranean), natural vegetation (Mediterranean‐type scrublands including phrygana and low maquis), as well as the pollinator fauna  the petals (white for humans); (b) the distal half of the filaments of the stamens (pink‐magenta); and (c) the green basal parts of the dorsal pair of petals providing access to the nectary . We employed the visual system of the tobacco hornworm hawkmoth (Manduca sexta L. [Sphingidae]), which has been extensively studied. Tobacco hornworms have three types of photoreceptors in the ventral part of their retina, which is considered as functionally specialized for foraging: UV ; blue (450 nm); and green (520 nm) . The Hymenoptera have a highly conserved trichromatic visual system showing maximal sensitivity at 344 nm (UV), 436 nm (blue), and 544 nm (green) . We used the well‐studied tetrachromatic visual system of the swallowtail butterfly Papilio xuthus L. (Papilionidae), showing maximal sensitivities at 360 nm (UV), 400 nm (violet), 460 nm (blue), 520 nm (green), and 600 nm (red)  in southern Spain and southern India . We used the visual system of Macroglossum stellatarum L. [Sphingidae] (hummingbird hawkmoth), which occurs in the study area, although it has not been observed on C. spinosa. This hawkmoth shows maximal sensitivities at 349 nm (UV), 440 nm (blue), and 521 nm (green)  the triangular spaces corresponding to the visual system of Chittka, , and (c)r) , here measured as the Euclidean distance between two loci in the bee hexagon. The terms Mo, K, and r are constants unique to the observer and conditions being modelled. For A. mellifera, Mo = 0.492, denoting the lower asymptote; K = 1, denotes the upper asymptote; and r = 78.5, denotes the increment rate. Similarly, a four‐parameter sigmoidal function . Additionally, we performed samplings at another two time points (repeated measures): night (01:00‐02:30), and dawn (05:30‐07:00) to examine temporal variability in scent emissions.−1 flow rate. The freshly opened flowers were enclosed in PET oven roasting bags with thickness of 12 μm  10 min prior to sampling. Adsorbent traps contained 10 mg of Porapak® Q , packed between two plugs of silane‐treated glass wool (Supelco) in a borosilicate glass Pasteur pipette (ø 7 mm). Additionally, during each sampling session, two ambient samples were collected from empty oven bags placed nearby to control for non‐floral contaminants.We employed dynamic headspace sampling, using PAS‐500 personal air samplers  set at 200 mL · minC. spinosa emanated from the accumulated pollen grains inside the used headspace bags, indicating that one source of floral scent must be the anthers. To explore the intrafloral spatial variation of scent emissions, we sampled separately the petals , stamens, and the calyx + gynoecium module , and nectar. This experiment was inspired by our initial observation that the intense scent of Appendix . We extrAppendix  and samp2, and 1 ng of toluene  was added as an internal standard to estimate emission rates (ER) in toluene equivalents per fresh mass of plant tissue . For the ER calculation we used the formula outlined by Svensson et al.  and acetone , recommended by Kaiser and Kraft  to optimn et al. .−1. The GC oven was held initially at 40°C for 3 min and the temperature was increased at 10°C min−1 to 250°C for 5 min. The two eluents (hexane and acetone) were tested for contaminants using the same method; apart from some other traces, diacetone alcohol (CAS: 123‐42‐2) was the only abundant contaminant.Scents were analysed on an Agilent 7890 A/5975 C GC/MS system  using splitless injections at 240°C on a polar column  and He as a carrier gas with a flow rate of 1 mL minhttps://webbook.nist.gov/). Whenever possible, we compared VOC retention times and mass spectra to those of authentic standards. Compounds were assigned to major biosynthetic classes based on the convention in the literature on floral volatiles  was used to retrieve the GC/MS data, and AMDIS version 2.62 software for peak deconvolution combined with NIST 05 Mass Spectral Library version 2.0d  to identify VOCs. Kováts Retention Indices were calculated for all the VOCs after analysis of an authentic alkane mix  under the abovementioned chromatographic conditions, using the non‐isothermal equation of van den Dool and Kratz , we applied non‐metric multidimensional scaling (Bray‐Curtis distance) on the quantitative dataset using the function C. spinosa scent . The separate statistical analyses of the subsets (biosynthetic pathways) is deemed more conservative than testing the entire dataset, as it will occupy fewer degrees of freedom in each model tested, reducing the dimensionality of the data. Moreover, recent studies have indicated the utility of grouping compounds by biosynthetic class, resulting from common patterns of correlated data due to shared biosynthetic pathway flux and ecological/ecophysiological functionality  of the R package mvabund version 4.1.12 , in which the response variable was the entire quantitative matrix of the volatile distribution across samples (log+1 transformed), and the independent variable was the floral part. In total, three models were built corresponding to each one of the major biosynthetic pathways of hclust.Based on the total quantitative scent composition, we ran a hierarchical cluster analysis to determine the affinities among the volatile emissions of the different floral parts sampled. For this, we used Euclidian dissimilarity, and the Ward's minimum variance clustering method in the R function To test the quantitative variation of emission rates among the different plant parts, we applied ANOVA for each one of the three major biosynthetic classes, followed by post hoc Tukey's range tests.C. spinosa from 20:00 to 10:00 on the next day , the sky was clear, and the moon was transitioning from the waning crescent to the waxing gibbous phase. Floral visitors were identified with visual inspection of the recorded material. For each visitor taxon, visitation was calculated as number of visits· flower−1 · hour−1.Hawkmoth visits are quick and can occur throughout the night, thus they can be easily missed. Therefore, motion‐activated night camera traps help to better understand nocturnal pollination Steen, . In JulyAppendix . In totaP = 0.002)  Figure . Moreove) Figure .Manduca sexta or Macroglossum stellatarum), in which all floral parts appeared equally color‐saturated. For the visual system of honeybees, the green part of the petals showed the highest degree of color saturation and differed significantly from the other flower parts. Finally, according to the visual system of swallowtail butterflies (P. xuthus), all floral parts showed equally low color saturation  are predicted to accurately (P ~ 1.0) discriminate between white vs. green petals, and green petals vs. stamens, but to choose at random between white petals and stamens (P = 0.46 ± 0.07). as the colors of these organs cannot be discriminated from one another.The models of correct color discrimination showed that honeybees are predicted to accurately discriminate all floral parts of r Figure . In contCapparis spinosa showed high floral volatile emission rates , in which the major biosynthetic classes were monoterpenes, nitrogenous compounds, and benzenoids. In total, we identified 59 VOCs (Table Cs Table .E)‐β‐ocimene represented >25% of the total emissions in both populations, followed by linalool , and methyl benzoate .The six identified nitrogenous aldoximes showed the highest combined emission rates . , benzenoids , and monoterpenes . The univariate post hoc tests identified several compounds that showed significant distribution patterns among the floral parts sampled and are shown in detail in Fig. Regarding intrafloral volatilomics, there was a clear variation among the different floral parts and nectar for all three major biosynthetic classes Figure . The MGLAccording to hierarchical clustering, the petals showed the lowest volatilomic affinity to the other floral parts or to nectar Figure  and emitF3,15 = 2.66, P = 0.086). Nitrogenous compounds were emitted at higher rates from the corolla and the stamens, compared with calyx + gynoecium and nectar . A similar pattern was found for monoterpenes , although in the nectar their concentration was higher than nitrogenous compounds.The highest floral emissions came from the anther‐bearing stamens and the corolla regarding all the major biosynthetic classes Figure . Overall−1 h−1), followed by dawn/morning (7.3 ± 1.7 visits fl−1 h−1). Nocturnal visits were observed only during three out of the six nights and were more infrequent than the other two time‐periods (0.2 ± 0.1 visits · fl−1 · h−1). The most frequent visits were by bees , which probed only for nectar, were potential pollinators since they swept pollen from the anthers with their large bodies and rough movements and could touch the stigma during a floral visit. In total, large bees showed an average visitation rate of one visit every 40 min per flower, either at dusk or after dawn.The highest overall visitation rate was observed around dusk, right at the beginning of anthesis  in three out of the six nights of observations in 2014 (Table Agrius convolvuli L. (Sphingidae), always visiting between ca. 23:00 and 05:00. Hawkmoths clearly probed for nectar using their proboscides  formed large aggregations on plants  , which, however, has a much longer anthesis time than our focal species ‐β‐ocimene, 3‐methyl‐butyraldoxime and 2‐methyl‐butyraldoxime, methyl benzoate, benzyl alcohol etc.), and the corolla shows the highest chemodiversity in relation to the other floral parts  , they can make up almost 50% of total emissions  flowers ; Raguso et al., Populus  trees .Interestingly, in Summers, , and of E)‐β‐ocimene and linalool also were abundant and spatially diffuse compounds in C. spinosa, including abundant representation in floral nectar  reduces oviposition by H. lineata, which represents a pollinating herbivore for this plant   Druce, C. odoratissima Jacq., C. buwaldae M. Jacobs)  Iltis,  and withC. spinosa represents a constant nectar resource for the vagrant hawkmoths crossing the Mediterranean Basin in the driest season of the year  showed an 8‐fold higher visitation rate than hawkmoths (Table Hyles lineata subsp. livornica Esper and Agrius convolvuli) are by far less frequent or entirely absent from many populations  were the principal floral visitors of C. spinosa and concluded that the only hawkmoth species observed (A. convolvuli) was not a pollinator because its body did not touch the anthers or stigma. We disagree with this conclusion for C. spinosa because hawkmoths can finely pollinate flowers with the pollen that they pick up with their proboscides, even in short‐tubed flowers   , the floral phenotype is clearly biased towards moths, particularly regarding the timing of anthesis and volatile emissions. Obviously, no “pro‐bee” chemical adaptations were necessary for establishing carpenter bee visitation  summertime flowering, and (b) nocturnal anthesis. Thus, it could be hypothesized that a phenotypic transition should not be necessary, if the metabolically costly floral display could be maintained, a fact that brings us to the second paradox, i.e., the over‐stated floral advertisement, with particularly high N‐emissions, coupled with copious nectar rewards, appears to target a putative pollinator class that is rare or inconsistent.Visitation to hawkmoth‐pollinated flowers by honeybees and carpenter bees is relatively common, both in the Mediterranean region  in the Rocky Mountains  in coastal California  in lowland environments in Japan  and water (for nectar). We suggest that the conspicuous but ephemeral flowers of C. spinosa are not resource‐limited, probably owing to two important ecophysiological adaptations that secure resource sufficiency. First, the plant fixes its own N2  floral scent is typically sphingophilous, (b) petals and anthers are the main sources of VOCs, and (c) sensory/ecological flexibility is implied by reflectance and color patterns. Our findings strongly suggest that C. spinosa has evolved in tight connection with hawkmoths, without evolving concomitant traits that exclude visitation by large bees.Although the flowers of C. spinosa evolved indeed in a (sub‐)tropical semi‐arid area, where nocturnal hawkmoths were key pollinating agents. Currently, the adaptive peak for C. spinosa would be regular pollination by large bees, enhanced by irregular pollination  by hawkmoths.Intriguingly, the observed inconsistency between flower visitation and phenotype has been maintained owing to the lack of resource limitation for water and nitrogen, representing a unique case in literature. We believe that it is plausible that C. spinosa is not simple to track. However, the genus Capparis provides a valuable case study of comparative floral ecology and evolution, given its vast geographical distribution and the multiple floral morphs and pollination systems represented therein, indicating a high degree of evolutionary plasticity.Given its ambiguities, the natural history of A.K. and T.P. conceived the idea; A.K. collected the data; A.K., J.E.G., R.A.R., A.G.D., R.S., and T.T. contributed analysis tools; A.K. and J.E.G. performed analyses; A.K. wrote the first draft; all authors contributed to subsequent drafts.Appendix S1. Figures S1–S5; Tables S1–S5.Click here for additional data file.Appendix S2. Video “Floral visitors of Capparis spinosa on Lesvos Island, Greece.”Click here for additional data file.


Previous epidemiology studies have primarily focused on stress in the home, rather than support, and whether psychosocial factors modify the association between pollution and health outcomes, rather than whether pollution exposure modifies associations between psychosocial factors and health outcomes.Outdoor air pollution, including ozone  ages 12–17 who reported an asthma diagnosis and lived within 5 mi of the nearest air monitoring station had linked O3 data for a 12-month period preceding the survey interview date. Adolescents reported perceived available support from an adult at home and frequency of asthma symptoms.Data from the cross-sectional 2003 representative, population-based California Health Interview Survey were linked with air quality monitoring data on O3 pollution regions, greater perceived support was related to lower asthma symptom frequency. Follow-up analyses suggested that the most plausible interpretation of the interaction was that O3 exposure modified the association between perceived support and symptom frequency. O3 × perceived support interactions were not statistically significant after adjusting for covariates.In unadjusted models, for adolescents living in high O3 exposure. Future work may benefit from incorporating personal pollution exposure assessments, comprehensive family environment assessments, and longitudinal follow-up of asthma exacerbations over time.These data provide preliminary evidence that the association between the lack of support in the home environment and worse asthma symptoms may be stronger in areas with higher O O3 was not related to asthma symptom frequency at ≤ 3 mi, B = − 0.13 , p = 0.29, or ≤ 5 mi, B = − 0.06 , p = 0.51.Higher OB = − 0.25 , p < 0.05; perceived support did not differ between the three highest poverty level groups. Older children reported greater symptom frequency, B = 0.10 , p = 0.042. However, symptom frequency and medication use were not related to sex, race/ethnicity, poverty level, or having insurance. Reporting using daily asthma medication use was related to higher symptom frequency, B = 0.67 , p < 0.001. The likelihood of having an asthma attack or visiting the ER/urgent care in the past year was not related to age, sex, poverty level, or insurance status. White adolescents were more likely to report an asthma attack in the past year, OR = 2.81 , p = 0.048, compared to Latino adolescents . Children in the American Indian/Asian/Pacific Islander/other category were less likely to visit the ER, OR = 0.23 , p = 0.009, compared to Latino adolescents (who did not differ from African American or White adolescents).To present associations among demographics, perceived support, and asthma outcomes, we focus on the ≤ 5 mi sample. Perceived support was not related to age, race/ethnicity, daily medication use, or having insurance. Adolescents from the lowest poverty level (0–99%) reported less perceived support compared to adolescents from the highest poverty level (> 300%), 3 and of perceived support on frequency of symptoms, and significant O3 × perceived support interactions were observed for participants living ≤ 3 and ≤ 5 mi of an air monitoring station but only in unadjusted models  was 0.06 (SE = 0.21), t = 0.31, p = 0.76; by comparison, in a high O3 exposure region (mean + 1 SD), the simple slope was − 0.51 (SE = 0.25), t = -2.04, p = 0.045. Figure 3 ≥ 48.8 ppb. However, after controlling for covariates, the O3 × perceived support interaction was in the same direction but no longer statistically significant (both ≤ 3 and ≤ 5 mi samples).When asthma symptom frequency was treated as a continuous variable, there were no main effects of Oon mean – SD was 03 and of perceived support on likelihood of intermittent vs. persistent symptoms in unadjusted and adjusted models . Figure 3 exposure. For example, in adolescents living in a high O3 exposure region (mean + 1 SD) in the ≤ 5 mi sample, the estimated probability of reporting persistent symptoms was 52.5%  for those reporting –1 SD perceived support  compared to 31.8%  for those reporting perceived support = 4 .Approximately 40% of the sample reported persistent asthma symptoms Table . Similar3 concentration or perceived support on the likelihood of an episode or attack, or ER/urgent care visits, or statistically significant O3 × perceived support interactions .In unadjusted and adjusted logistic regressions with the ≤ 3 and ≤ 5 mi samples, there were no main effects of O3 exposure greater than 48.8 ppb, reporting more perceived support at home was related to lower asthma symptom frequency, and reporting less perceived support at home was related to greater risk of persistent asthma symptoms. While our analyses suggested that O3 exposure modified the association between perceived support and asthma symptoms, rather than perceived support buffering against the effects of O3 exposure, after adjusting for covariates, the O3 × perceived support interaction was no longer statistically significant. While associations with intermittent vs. persistent symptoms were independent of SES (% poverty level) and daily prescription asthma medication use, the same pattern did not extend to reporting an asthma attack or a visit to the ER or urgent care because of asthma in the past year. Prior studies focused on stress in the family environment [As adolescents become more independent, knowing that adults/parents are available when needed takes on increasing importance . In unadironment , 18; whi3 × perceived support effect.Two primary reasons for considering the reported findings preliminary are the differences between unadjusted and adjusted models and the cross-sectional study design. In adjusted models, poverty level was the only covariate systematically related to perceived support and was not systematically related to symptom frequency. In contrast, several covariates showed relatively strong associations with symptom frequency, age, Latino or African American ethnicity, and taking daily prescription control medication, with the latter two related to lower symptom frequency. Notably, other analyses of CHIS data found higher likelihood of daily asthma medication use among Latino and African American children , 33. Thu3 interacts with molecules in the airway to stimulate innate inflammatory responses from lung macrophages and dendritic cells [These preliminary, cross-sectional data suggest several plausible interpretations that should be explored in future prospective longitudinal work. First, by increasing airway inflammation, pollution may also increase neural sensitivity to social experiences, which may further increase systemic and airway inflammation contributing to symptom severity. Oic cells . Experimic cells , includiic cells  and heigic cells . Heighteic cells  and, in ic cells . Innate ic cells . Furtheric cells , 37 and ic cells , 38.Another possible interpretation is even though increased symptoms may occur because of high pollution exposure, a supportive home environment may alleviate the effects due to better adherence to medication and other treatment regimens. In cross-sectional data, greater family conflict or stress is related to lower adherence ; less is3 rather than pollutants with high spatial variability like NOx and particulate matter, where exposure modeling of multiple variables  enables estimating pollutant levels at a specific geographic location  [3 has less spatial variation compared to NOx, particularly in rural areas [3 exposure are generally similar across types of exposure assessment and spatial concentration of air monitors [3 and asthma symptoms is small  , 18, 26.xposures , a critixposures –7. Simil3 levels, which may be explained by two observations in the literature. First, O3 concentrations are higher in suburban and rural areas that are downwind of air pollution sources, which are also higher income relative to urban areas [Regarding generalizability, because of requirements related to living within 5 mi of an air monitoring station, the subsample of adolescents with asthma in CHIS is small, underpowered for detecting small effects, and may not be representative of adolescents with asthma in California or other regions. In our sample, greater perceived support showed a small to modest association with higher Oan areas . Second,an areas  and greaan areas .The observed findings are consistent with models that integrate psychosocial and pollution exposures to explain poor outcomes in at-risk populations –7, 9. Fu


Ionic liquids (ILs) loading ketoconazole (KCZ) have shown better efficacy on rats with tinea pedis than the marketed Daktarin® but clinical studies are still lacking. In this study, we described the clinical translation of ILs containing KCZ (KCZ‐ILs) from the lab into the clinic and evaluated the efficacy and safety of KCZ‐ILs in patients with tinea pedis. Thirty‐six enrolled participants were randomized to receive either KCZ‐ILs  or Daktarin®  topically twice daily, making the lesion be covered with a thin layer of medication. The randomized controlled trial lasted for 8 weeks including 4 weeks of intervention and 4 weeks of follow‐up. Primary efficacy outcome was the proportion of treatment success responders, defined as patients achieving negative mycological result and ≥60% relative reduction in total clinical symptom score (TSS) from baseline at week 4. Secondary outcomes mainly for evaluating the relapse of disease included the proportion of treatment success individuals at week 8 and fungal recurrence rate at weeks 2, 3, 4, and 8. After 4 weeks of medication, 47.06% of the KCZ‐ILs subjects were treatment successes compared with only 25.00% of those using Daktarin®. Throughout the trial period, KCZ‐ILs induced a significantly lower recurrence rate (52.94%) than that of control patients (68.75%). Furthermore, KCZ‐ILs were found to be safe and well‐tolerated. In conclusion, ILs loading only 1/4 KCZ dose of Daktarin® showed a better efficacy and safety profile in the management of tinea pedis, creating a new opportunity for the treatment of skin diseases caused by fungal infection and is worthy of clinical application. Interdigitale, a major aetiological dermatophyte of tinea pedis, as well as a significant synergistic antimicrobial effect with KCZ.Ionic liquids (ILs) hold great promise to be optimal active pharmaceutical ingredients or drug carriers owing to enhanced drug solubility, bioavailability, and superior antifungal efficacy.Translation of the formulations from bench to bedside is of great significance, which could be facilitated by scale‐up, characterization, stability, mode of action, dosing, preclinical toxicology in animals, and finally, human studies on efficacy and safety.22.11H‐NMR analysis  to reflect the signature of KCZ‐ILs. After 14 months, KCZ‐ILs demonstrated minimal change in stability with 2.9% loss of KCZ and 3.6% loss of geranic acid, respectively Figure . Althoug2, followed by those of 1/4 KCZ‐ILs (1.67 ± 0.10 μg/cm2) and 1/16 KCZ‐ILs (0.74 ± 0.09 μg/cm2) groups. Notably, the differences between the KCZ‐ILs, 1/16 KCZ‐ILs groups and the control group (1.55 ± 0.15 μg/cm2) were significant (p < 0.0001) while the 1/4 KCZ‐ILs group and the control group demonstrated evenly matched effect. In consideration of the low adhesion of aqueous solution, the KCZ‐ILs that facilitated the penetration of KCZ into the skin by over twofold compared to the control group were selected for administration.Dosing was determined by the in vitro permeation test. The permeability of KCZ in ILs‐based aqueous solutions was assessed to determine the optimal formulation for the following clinical study. Among them, 1/4 and 1/16 KCZ‐ILs were obtained by diluting the KCZ‐ILs with water 4 and 16 times, respectively. As shown in Figure 2.2A total of 39 patients underwent screening and 36 satisfying inclusion and exclusion criteria were enrolled. Three patients were lost in follow‐up because of their job changes or unpunctuality during the study period. Of the 33 complete evaluable patients, 17 were treated with KCZ‐ILs and 16 with Daktarin®. The CONSORT diagram of the participants is given in Figure Among the 17 cases in the experimental group, there were nine males and eight females with the age of 31–59 years old, and the average total clinical symptom score (TSS) of 8.76 . In the control group, there were eight males and eight females, whose ages range from 32 to 59 years old, and their average TSS was 9.44 . A few patients  had associated comorbidities such as hypertension , diabetes , or both . Besides the body‐mass index, the characteristics of the patients at baseline did not differ significantly between the two groups (Table 2.3p < 0.001) in the KCZ‐ILs group and −4.44 (2.34) (p < 0.001) in the Daktarin® group. The symptoms began to improve after only 1 week of treatment, and the effect lasted for the whole study period which could be visualized directly from representative photographs of patients with TSS ranged from 8 to 12 , mainly contributed by weeks 2 and 3 (Table The change in TSS after 4 weeks of treatment was −4.94 (2.51)  in week 1, 64.71% (p < 0.05) in weeks 2–3. At the end of treatment, the clearance rate reached 76.47% with only four positive patients left. In contrast, the fungi clearance rate of Daktarin® remained the same at 50.00% in weeks 1–3 and reached 56.25% in week 4.Mycological eradication rates evaluated by direct microscopic examination were presented in Table Fungal recurrence cases appeared from the second week of dosing. The conversion rate of mycological negative to positive was the lowest at 9.09% at the third week of KCZ‐ILs dosing, and increased either by continuing the treatment for 1 week or by stopping it. In the control group, negative conversion rate remained at 37.5% from the second week to the fourth week of treatment, and unexpectedly dropped slightly after discontinuation. Throughout the trial period, there were nine (52.94%) fungal negative cases changed to be positive for KCZ‐ILs, compared with 11 (68.75%) of 16 for Daktarin®.2.5p = 0.010) in favor of KCZ‐ILs at week 4. Secondary efficacy outcomes were mainly for evaluating the relapse of disease. The proportion of treatment success individuals at endpoint, 4 weeks post‐treatment, was 41.17% for KCZ‐ILs compared to 31.25% for control subjects.As the primary efficacy variable, overall efficacy was derived by integrating with mycological results and TSS improvement. A four‐grade scale  with proper modification of previously published categories was used in this study.2.6No patients applying the KCZ‐ILs to lesion sites reported adverse reactions. One patient (6.25%) in the control group experienced transient mild erythema in the lesions 1 week after application of the drug, which did not affect the continuation of treatment and the determination of efficacy.3Clinical trials are conducted in humans to test new treatments, which are typically evaluated over several phases to determine their efficacy and safety. Phase 1 trials focus on the safety and tolerability, generally with a small number of healthy participants . Phase 2 trials are the first time that new treatments are taken in target patients to observe the preliminary efficacy and safety, and to assess whether they have meaningful benefits to warrant further investigation in phase 3 trials. As phase 3 trials are usually multicenter, large‐scale, and long‐period trials, phase 2 trials acting as pioneers are of great significance.ILs have shown potential for applications as antimicrobials. The choline‐based ILs, especially those synthesized with choline bicarbonate and geranic acid at ratios of 1:1 and 1:2, have been reported to possess prominent skin permeability and antimicrobial activity.p < 0.001) and control groups . Both formulations began to relieve the symptoms remarkably after only 1 week of dosing, and the effect lasted for the whole trial period. In particularly, the curative effect in KCZ‐ILs group was significantly better than that in the control group at weeks 2 and 3 (p < 0.05), meaning KCZ‐ILs exerted a more powerful effect. Further analysis on each sign showed that significant declines (p < 0.0001) of the severity were observed in lesion area, scaling, keratinization, and pruritus for KCZ‐ILs, as well as scaling and pruritus for Daktarin®. It reveals that the reason for the superiority on TSS improvement of KCZ‐ILs over Daktarin® may lie in the alleviation of lesion area and keratinization.TSS was computed as the sum of seven signs attributable to tinea pedis at the lesion site where each sign score was rated on a scale of 0 (not present) to 3 (severe).Mycological cure was generally defined as negative potassium hydroxide (KOH) examination findings and negative culture results.Overall efficacy was finally evaluated according to a four‐grade scale  derived from TSS changes and mycological results.Relapse is a result of the re‐appearance of the same original strain, which is easy to occur in many diseases and can be influenced by detection methods of post‐treatment testing.In terms of safety, one patient in the control group experienced transient mild erythema, but none discontinued the treatment. Meanwhile, KCZ‐ILs were found to be safe and well‐tolerated.The limitations of this study include the small sample size and short follow‐up period. Thus, long‐term maintenance of efficacy and long‐term adverse events could not be assessed.4With reference to phase 2 trials, this study was designed to be a randomized controlled clinical trial with a small sample size, assessing the efficacy and safety of a new ILs‐based formulation named KCZ‐ILs. The results showed that KCZ‐ILs  showed significant superiority than Daktarin®  in both clinical improvement and relapse prevention. Besides, KCZ‐ILs were very well‐tolerated, without any adverse event while one patient in the control group reported transient mild erythema. In conclusion, ILs loaded only 1/4 KCZ dose of Daktarin® showed a better efficacy and safety profile in the management of tinea pedis, which creates an effective pharmaceutical formulation for the treatment of skin diseases caused by fungal infection and is worthy of clinical application.55.1KCZ‐ILs were specifically KCZ‐loaded [Ch][Ger] aqueous solution where [Ch][Ger] was synthesized by choline bicarbonate and geranic acid at a molar ratio of 1:1. Firstly, a mixture of [Ch][Ger]/Tween‐80/glycerin/KCZ was prepared in the way reported previously.KCZ‐ILs were characterized by FT‐IR spectra and DSL. The stability of KCZ‐ILs in brown glass bottles was evaluated at room temperature over a 14‐month period. The representative components (KCZ and geranic acid) in KCZ‐ILs were monitored and the degradation percentage of KCZ was determined using HPLC.2 and a receptor volume of 7.8 ml. Phosphate buffered saline (pH 7.4) containing 20% ethonol (v/v) was used as the receptor medium, which was stirred at 300 rpm, 37°C. Test and control formulations of 500 μg were applied to the stratum corneum side of the skin samples for 6 h, respectively. To extract as much of KCZ as possible, the skin was washed and horizontally cut into pieces (20 μm). The layers of one skin sample were collected in a tube containing 480 μl of methanol and 20 μl of the internal standard bifonazole (504 μg/ml), followed by ultrasonic for 30 min and centrifuged at 20,000 rpm for 5 min. The amount of KCZ in the supernatant was measured using HPLC.Dosing of formulations was determined by the in vitro permeation test. Specifically, the porcine skin samples, harvested from 2 weeks old domestic pig, were mounted onto standard Franz cells. The cells had a contact area of 1.77 cm5.2This randomized clinical trial was conducted at Shanghai skin disease hospital in China from August 1, 2021 to July 31, 2022. It is approved by the institutional ethics committee and registered on the Chinese Clinical Trials Registry (ChiCTR2000035486). Given the treatment success rate by week 4 of 90% for KCZ‐ILs and 41% for Daktarin® in the pre‐experiment, 36 patients (18 in each arm) were required, to achieve 90% power with an alpha of 0.05, accounting for 20% dropout. A total of 39 consecutive patients diagnosed with tinea pedis were screened and 36 satisfying inclusion and exclusion criteria were enrolled. Random number sequence was generated on computer by a simple randomization schedule. Each randomization number was sealed in an opaque envelope with a serial number on it. After signed a written informed consent, the patient with a known serial number opened the corresponding envelope to be informed of the assignment. All the patients were randomized (1:1) to receive either KCZ‐ILs or Daktarin®. Clinic visits occurred on week 0 (baseline), during weeks 1, 2, 3, and 4 (the end of the treatment period), and at week 8 (4 weeks after intervention). Compliance was assessed via the daily patient diary and the dosage was monitored by means of formulations weighing. Patients applying 80 ~ 120% of the expected doses at required time were considered to be compliant. To reduce the risk of bias, the dermatologists, mycelial examiners, data collectors, and statisticians were blinded to the information of patients until the data were locked. Changes in TSS, mycological eradication rates, and adverse effects were recorded at each visit.5.3With reference to the 2017 edition of Guidelines for the Diagnosis and Treatment of Tinea Manuum and Tinea Pedis, tinea pedis is clinically classified into blistering, interdigital erosion, and scaly keratosis based on the morphology of the lesions. Patients aged between 20 and 65 years, with any type of tinea pedis and positive fungal microscopy at the lesion site, as well as good consciousness were included. Patients were excluded if they were receiving any medication within 1 month of screening, allergic to the drugs in this study, or pregnant/lactating women. Those who withdrew in the middle of the study, or changed drugs without authorization were also excluded.5.4KCZ‐ILs  prepared as described above was used in the experiment group while the commercial Daktarin®  was adopted in the control group. After cleaning the identified lesions, the patients topically self‐applied the test product using a cotton swab applicator, where the applied amount was able to make the lesions be covered with a thin layer of medication. The product was applied topically twice daily for 4 weeks.5.5The primary efficacy outcome was the proportion of treatment success responders,Secondary outcomes mainly for evaluating the relapse of disease included the proportion of treatment success individuals at week 8 and fungal recurrence rate at weeks 2, 3, 4, and 8. Besides, TSS changes, mycological eradication rates, and the compliance at each visit were analyzed. Safety was assessed by physical examination, vital signs, and adverse events, especially irritation based on erythema, peeling, papules, and blisters.5.6t‐test for normally distributed data and Mann–Whitney test for skewed/ordinal data. Qualitative or categorical variables were described by case number and proportions No. (%). Proportions were compared via Chi‐square, McNemar or Fisher's exact test, whichever was the most applicable. All statistical tests were two‐sided and performed by Statistical Package for Social Sciences (IBM SPSS Statistics 26) at a significance level of p < 0.05.Efficacy analyses were based on the per‐protocol population, which comprised the patients who had completed the entire trial as prescribed after randomization. All quantitative variables are represented as mean and standard deviation (SD). The means were compared by Student's Xiying Wu: Data curation ; funding acquisition (lead); project administration (lead); supervision ; validation ; writing – original draft (lead); writing – review and editing . Min Shen: Data curation ; project administration ; resources . Huan Wang: Data curation ; funding acquisition ; writing – original draft ; writing – review and editing . Xue He: Data curation ; project administration . Jingwen Tan: Methodology ; project administration . Ruiping Wang: Methodology (supporting). Lianjuan Yang: Conceptualization (supporting); project administration (supporting). Hong Yang: Project administration (supporting). Jianping Qi: Conceptualization ; writing – review and editing (lead). Zhongjian Chen: Conceptualization ; resources (lead); supervision . Quangang Zhu: Conceptualization ; funding acquisition ; supervision (lead); writing – review and editing .The authors have no conflicts of interest relevant to this article.https://publons.com/publon/10.1002/btm2.10463.The peer review history for this article is available at Appendix S1. Supporting Information.Click here for additional data file.


Capillary blood was collected onto both conventional newborn screening (NBS) cards and VBCDs. Specimens were analysed by liquid‐chromatography tandem mass‐spectrometry (LC–MS/MS) using a common calibrator. Use of VBCDs was evaluated qualitatively by patients. Mean bias between plasma and volumetrically collected capillary DBS Phe was −13%. Mean recovery (SD) of Phe from DBS was 89.4% (4.6). VBCDs confirmed that the volume of blood typically assumed to be present in a 3.2 mm sub‐punch is over‐estimated by 9.7%. Determination of the relationship between plasma and capillary DBS Phe, using a single analytical method, common calibration and VBCDs, demonstrated that once the under‐recovery of Phe from DBS has been taken into account, there is no significant difference in the concentration of Phe in plasma and capillary blood. Conversely, comparison of plasma Phe with capillary DBS Phe collected on a NBS card highlighted the limitations of this approach. Introducing VBCDs for the routine monitoring of patients with PKU would provide a simple, acceptable specimen collection technique that ensures consistent sample quality and produces accurate and precise blood Phe results which are interchangeable with plasma Phe.Measurement of plasma and dried blood spot (DBS) phenylalanine (Phe) is key to monitoring patients with phenylketonuria (PKU). The relationship between plasma and capillary DBS Phe concentrations has been investigated previously, however, differences in methodology, calibration approach and assumptions about the volume of blood in a DBS sub‐punch has complicated this. Volumetric blood collection devices (VBCDs) provide an opportunity to re‐evaluate this relationship. Paired venous and capillary samples were collected from patients with PKU ( Volumetric blood collection devices provide a simple, acceptable specimen collection technique that ensures consistent sample quality and produces accurate and precise blood Phe results which are interchangeable with plasma Phe.1Accurate and reproducible measurement of phenylalanine (Phe) is key to the management of patients with phenylketonuria ; the 2017 European guidelines for diagnosis and management of patients with PKU recommend consensus age‐related blood phenylalanine (Phe) target treatment ranges to prevent adverse neurological outcomes and specify three clinical decision points .n = 310) and DBS (n = 95) was 9.8% and 20.1% respectively.Data from the 2022 European Research Network for the evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDIM) Quantitative Amino Acids External Quality Assessment (EQA) scheme and Special Assays in Dried Blood Spots (SADBS) EQA scheme show that the mean inter‐laboratory relative standard deviation (%RSD) for Phe in plasma  and the non‐volumetric conventional NBS collection card routinely used for PKU monitoring. Analysis by LC–MS/MS and quantification against a common calibrant compensated for measurement bias due to methodology, calibration or standardisation. Likewise, the use of VBCDs negated differences due to DBS specimen quality, volume and haematocrit of the blood applied to the filter paper and/or assumed volume of blood in a sub‐punch from a DBS specimen.2Full details of the materials and methods used are provided in Appendix 2.1n = 51) were provided with information sheets and gave informed consent. Three paired samples were collected from each patient during a routine clinic visit; venous blood (5 mL lithium heparin); capillary blood on a Capitainer‐qDBS VBCD (2× 10 μL); capillary blood on a filter paper collection device (2 drops). Immediately post‐collection the venous blood was centrifuged and plasma removed and stored at −20°C until analysis. Capillary blood samples were dried at ambient temperature for 3 h then stored in foil bags with desiccant at −20°C. The quality of the DBS specimens was assessed against the proposed UK Metabolic Biochemistry Network guidelines for monitoring of inherited metabolic diseases.The study was reviewed by local research and development departments and approved as a service evaluation; formal ethical approval was not required. Patients . The 95% confidence intervals (CI) for the slope and intercept were 0.8598–0.9130 and −17.31 to 5.042 respectively, indicating a proportional difference between plasma and capillary Phe but no systematic error  and the residual standard deviation (95% CI) was 45.5 (−89.2 to 89.2). Bland–Altman analysis showed a mean bias of −13.3% (95% limits of agreement (LoA) −29.03 to 2.44%) . The 95% CI for the slope and intercept were 1.019–1.178 and −20.15 to 19.31 respectively, indicating a proportional difference between volumetric and non‐volumetric capillary DBS but no systematic error. The cusum test confirmed no significant deviation from linearity (p = 0.6862). The RSD  was 71.61083 (−140.3522 to 140.3522). Bland–Altman analysis showed a mean bias of 9.42% (95% LoA −14.32 to 33.17%).Plasma Phe concentrations in the patient specimens ranged from 52 to 1899 μmol/L . Passing–Bablok regression equation for plasma versus capillary phenylalanine collected with VBCDs was r Figure . The cus) Figure . PassingThe results of the qualitative evaluation of patient satisfaction with the Capitainer‐qDBS are summarised in Figure 4Although traditionally imprecision is often deemed acceptable when <15%,y = 0.9981x + 3.0304, R2 = 0.9838.The results show that the mean bias between plasma and capillary blood Phe using VBCDs is −13%. The use of a common analytical method, calibration and operator excludes method and/or calibration bias as contributing to this difference, a key differentiator from previously reported studies.n = 20).An alternative explanation is that there are differences in the distribution of Phe between plasma and erythrocytes/blood. However, it has been demonstrated previouslyTraceCERT®). By implementing these changes, laboratories could prevent misinterpretation of Phe concentrations derived from different specimen types. This would be especially useful in paediatric and pregnant patients, where treatment targets have been linked explicitly with clinical outcomes such as neurological function in paediatric patients, and maternal PKU syndrome in pregnant individuals respectively.Establishing that there is no physiological difference between plasma and capillary DBS Phe means laboratories can routinely use an aqueous calibration to measure both plasma and capillary DBS Phe, provided they account for the recovery of Phe from the DBS and collect the specimens with a VBCD. This is advantageous as it provides a common point of reference within a given laboratory and importantly, between laboratories; a certified reference material for Phe in aqueous solution being commercially available  =  × (concentration of SIL) × (150/3.1). Hence, if the volume of blood in a 3.2 mm sub‐punch was overestimated, and is in fact 2.8 μL, the error introduced would result in the “true” Phe concentration being underestimated, that is, the difference between (150/3.1) and (150/2.8) equates to (53.57–48.39)/53.57, equivalent to a difference of −9.7%.Determining the exact volume of blood present in a 3.2 mm sub‐punch of a conventional DBS is not the intended aim of this study. Experimental determination of this parameter is complexIntroducing VBCDs would potentially solve this problem and, whilst it is acknowledged that the Captainer qDBS was evaluated by a small number of patients, the results indicate that the device would likely be well received by this patient group. However, the device costs approximately £2.50 more than the conventional NBS card. Based on the number of patients on diet in England, the recommended frequency of testing5Implementing VBCDs for the routine monitoring of blood Phe will ensure better quality specimens by negating issues relating to DBS size, quality and haematocrit. This should be considered especially in patient subgroups where improved precision and accuracy of results, and avoidance of sample rejection could improve clinical care such as in children, pregnant woman and during sapropterin responsiveness testing. This study emphasises the need for clinical and laboratory health care professionals to better understand the true relationship between plasma and capillary DBS Phe, acknowledge the limitations of the current methodologies and translate this into clinical practice to ensure correct interpretation of laboratory tests and appropriate patient management. It would be advantageous to revise the 2017 guidelines using evidence from studies where appropriate scrutiny has been given to the analytical performance of the method used. The potential application of VBCDs to monitor other inherited metabolic disorders, for example, tyrosinemia type 1, maple syrup urine disease or classical homocystinuria, should also be investigated.Rachel S. Carling: conceptualisation, methodology, formal analysis, supervision, writing—original draft preparation. Zoe Barclay: investigation, validation, formal analysis, writing—review and editing. Nathan Cantley, Sarah L. Hogg, Yael Finezilber, Elaine Murphy, and Danja Schulenburg‐Brand: resources, writing—review and editing. Erin C. Emmett: writing—review and editing. Stuart J. Moat: validation, writing—original draft preparation.Rachel Carling, Zoe Barclay, Nathan Cantley, Erin Emmett, Sarah Hogg, Yael Finezilber, Danja Schulenburg‐Brand, Laine Murphy and Stuart Moat declare that they have no conflict of interest.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study.Appendix S1: Supplementary InformationClick here for additional data file.Appendix S2: Supplementary InformationClick here for additional data file.Appendix S3: Supplementary InformationClick here for additional data file.


Escherichia coli chaperonins GroEL and GroES (GroE), for which in vivo obligate substrates have been identified by proteome‐wide experiments. These substrates comprise various proteins but exhibit remarkable structural features. They include a number of α/β proteins, particularly those adopting the TIM β/α barrel fold. This observation led us to speculate that GroE obligate substrates share a structural motif. Based on this hypothesis, we exhaustively compared substrate structures with the MICAN alignment tool, which detects common structural patterns while ignoring the connectivity or orientation of secondary structural elements. We selected four (or five) substructures with hydrophobic indices that were mostly included in substrates and excluded in others, and developed a GroE obligate substrate discriminator. The substructures are structurally similar and superimposable on the 2‐layer 2α4β sandwich, the most popular protein substructure, implying that targeting this structural pattern is a useful strategy for GroE to assist numerous proteins. Seventeen false positives predicted by our methods were experimentally examined using GroE‐depleted cells, and 9 proteins were confirmed to be novel GroE obligate substrates. Together, these results demonstrate the utility of our common substructure hypothesis and prediction method.Molecular chaperones are indispensable proteins that assist the folding of aggregation‐prone proteins into their functional native states, thereby maintaining organized cellular systems. Two of the best‐characterized chaperones are the  Chaperonin GroE prefers proteins adopting α/β class structures as substrates. We assumed the common features in substrates by focusing on the local structures most exclusively included in substrates, and developed a GroE substrate predictor. The selected substructures are projectable onto a 2‐layer 2α4β sandwich, the most ubiquitous substructure in proteins, which explains the versatile role of GroE in folding. Nr. Nr2jjqAtes Fig.  25, 26]2jjqA  with at least one other SSE.MICAN with each mode provides the best local alignment of two structures, and thus we can obtain two aligned substructures. Among the products, we first selected the candidates of common substructures (loops excluded) satisfying the three criteria below.With the SQ, RW, and RR modes, 694, 928, and 1252 substructures were obtained, respectively.Next, the selected substructures were clustered by MICAN at each mode, to remove the redundancy. For two substructures aligned by MICAN, we calculated the alignment coverage . We applied the average‐linkage clustering, employing ‘1—the alignment coverage’ as the distance. The clustering terminated at a distance of 0.2 (0.8 coverage), and 203, 284, and 370 structural clusters were, respectively, obtained by the SQ, RW, and RR modes of MICAN. The closest substructure to the cluster centroid was regarded as the representative structure that defines the backbone of the KD template (below).To characterize the structural clusters in more detail, we considered the hydrophobicity patterns. The Kyte and Doolittle (KD) index  was emplMICAN estimated whether a KDT was found in a structure of a positive or negative sample, by applying structural and hydrophobicity criteria. First, the structures of a KDT and a positive or negative sample were compared by MICAN, using the same comparison mode as in the substructure selection. For each KDT and sample pair, the 5 best alignments were considered. We regard two structures as matched if more than or equal to 50% residues of all SSEs in the KDT were aligned, and the Cα RMSD was less than 3 Å (structure criterion). Next, for the alignments that satisfied the structure criterion, the hydrophobic pattern was evaluated. The distance (1‐NIP) of hydrophobic patterns between the aligned regions of the KDT and the sample should be within the cut‐off (hydrophobic criterion). We arranged all structures in the positive and negative samples from the left to right horizontally, and all KDTs were along the left side vertically. When a KDT was included in a sample structure, we placed a dot at the intersecting point Fig. . The illIn the development of a predictor using a union of KDTs, we exhaustively scanned the structure comparison mode and parameter space and tried to find their combination that defines the best predictor. When we constructed a structure‐KDT matching profile table, we specified the structure comparison mode , and the hydrophobic pattern matching cut‐off [threshold of ‘1‐NIP’ from rough (2) to strict (0)]. The constructed profile was examined for its ability to discriminate the positive and negative samples. We employed the jack‐knife test to estimate the performance, in which one column of data corresponding to one structure  were cloned downstream of the ous work . Each gein vivo was performed according to the previous work [m diaminopimelate and 0.2% glucose for depletion (GroE– conditions) or 0.2% arabinose as a control (GroE+ conditions). Each candidate protein was expressed under leaky conditions (without IPTG induction) during cultivation. After 3 h of cultivation, the cells were harvested, suspended in lysis buffer , and disrupted by sonication. After the disruption, the uncentrifuged total fraction and the supernatant fraction obtained by 20 000 g centrifugation were separated by SDS/PAGE. The proteins in each fraction were detected by immunoblotting with an anti‐HA monoclonal antibody  and an anti‐mouse antibody conjugated with horseradish peroxidase . The chemiluminescence signal was detected by a LAS4000 image analyzer (Fujifilm).The experimental evaluation of GroE dependency ous work . MGM100,The authors declare no conflict of interest.SM, TN, HT, and MO designed the project. SM, RK, and MO performed computations. TN and EU performed experiments. All authors analyzed data. SM, HT, and MO wrote the paper with input from all authors.Data S1. Alignments of positive samples and KDT1.Click here for additional data file.Data S2. Alignments of positive samples and KDT2.Click here for additional data file.Data S3. Alignments of positive samples and KDT3.Click here for additional data file.Data S4. Alignments of positive samples and KDT4.Click here for additional data file.Table S1. List of proteins and prediction results. Explanations of columns and symbols: Synonyms, –: no synonyms in UniProt; Sol and Sol (GroE), solubilities under GroE absent and present conditions, respectively; ΔSol, Sol (GroE)‐Sol; Class (Kerner), classes defined by Kerner et al. –: no class was defined; Class , classes defined by Fujiwara et al. and Niwa et al., 4: class 4 defined by Fujiwara et al., +: class 4 defined by Niwa et al., –: no class was defined; positive/negative/neutral, P: positive, N: negative, –: neutral; RW, prediction of the RW model; x: positive , –: negative; KDT1 ~ KDT4; hit of each KDT; x: positive, –: negative; RWRR, prediction of the RWRR model; x: positive (at least one of KDTs 5–9 hit), –: negative; KDT5 ~ KDT9; hit of each KDT; x: positive, –: negative; Structure, structure used for the prediction, Nat: native structure, Hom: homology model; SCCS, SCOP concise classification strings.Table S2. Performance of predictors.Table S3. Results of the best‐hit method.Table S4. KDTs used in the RW predictor. * Template was defined by the common substructure of JW1 and JW2. Their SCCSs are denoted in the SCCS 1 and 2 columns. ** Red numbers: positions of α helices.Table S5. Alignment of KDTs in the RW predictor and structure of positive samples.Table S6. KDTs used in the RW + RR predictor. * Template was defined by the common substructure of JW1 and JW2. Their SCCSs are denoted in the SCCS 1 and 2 columns. ** Red numbers: positions of α helices.Table S7. List of primers.Table S8. Performance of new single KDT. This table presents only the KDTs identifying positives that the RW and the RW + RR predictors could not detect. We selected the KDTs with one or no false positive. newTP (RW): number of true positive that the RW predictor could not detect. newTP : number of true positives that the RW and the RW + RR predictors could not detect. JW: ID of newTP . MCC: MCC of single KDT. MCC (RW): MCC of the RW predictor plus the single KDT.Click here for additional data file.Appendix S1. Additional information for results.Fig. S1. Results of hydrophobic criterion. The match of hydrophobic pattern (1‐NIP) and the averaged KD index for the aligned regions are plotted. (a) Positive samples. (b) Negative samples. Circles and crosses are the plots of alignments that passed the hydrophobic criterion and that did not, respectively. Plot of alignments against KDT1, 2, 3, and 4 are colored orange, green, red, and blue, respectively. Diamonds are the plots for KDTs, colored in the same manner to the alignments.Fig. S2. KDTs used in the RW + RR predictor.Fig. S3. Role of KDTs used in the RW + RR predictor. (a) Five‐circle Venn diagram showing how many positive samples (labeled by SCCS) are detected by each KDT used in the RW + RR predictor. SCCSs are shown in black characters. Multiple hits are denoted in parentheses. Red numbers are the number of positives in each box. (b) Similarity of the roles of KDTs. The similarity of two KDTs was estimated by the preferences in the positive‐sample detection using the Jaccard index. The roles of KDTs are divided into the main TIM (c.1) predictor, the supportive TIM predictor, the α/β protein predictor, and the outlier predictor.Fig. S4. Positive prediction rate by the RW + RR predictor against ΔSolFig. S5. Hit regions between RumB, BioC, and KDTs. RumB and BioC adopt the same fold (c. 66), and they are alignable. MICAN aligned both proteins. KDTs 3 and 4 hit RumB, and KDT7 hit BioC. Note that KDTs are only composed of SSEs. α helices and β strands are colored orange and green, respectively.Fig. S6. Evaluation of GroE dependencies in vivo for four proteins adopting TIM β/α barrel fold in the negative samples . Each protein was expressed under GroE+ and GroE‐ conditions and analyzed by a centrifugation‐based method (see in vivo.thod see . ‘T’ andFig. S7. Match regions of substrates adopting the TIM β/α barrel fold with KDTs 5 and 6. KDT5 and KDT6 identified 18 and 7 TIM β/α barrels, respectively. Each structure aligned to the reference structure match of each residue against KDTs was counted (see the legend of Fig. Click here for additional data file.


Adjuvant therapy for stage IB non‐small cell lung cancer remains debatable. In this real‐world study, we evaluate the efficacy and safety of adjuvant epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs) for resected stage IB lung adenocarcinoma.This real‐world study recruited 249 patients diagnosed with stage IB disease after surgical resection between January 2013 and September 2021. Sixty‐six (26.5%) patients received adjuvant targeted therapy (TKIs group), and 183 (73.5%) were enrolled in the clinical observation (CO) group. Propensity scores were matched to minimize the observed confounder effects between the two groups, and 59 patient pairs were matched. The primary endpoint was disease‐free survival (DFS).p = 0.008). DFS differences were found in the entire cohort (p = 0.005) and the matched cohort (p = 0.024) between the two groups. Multivariate analysis showed that adjuvant EGFR‐TKIs was an independent factor for DFS , along with poor cell differentiation , and spread through air spaces . None of the patients discontinued EGFR‐TKIs owing to the low occurrence rate of treatment‐related serious adverse events.In the TKI group, 38 (64.4%) patients chose to receive icotinib, 27.1% (16/59) received gefitinib, and 5 patients (8.5%) chose osimertinib. The median follow‐up time was 30.8 months (range: 7–107 months). Two (3.4%) patients in the TKI group and 10 (16.9%) in the CO group experienced disease relapse. The 3‐year DFS rates were 98.3% in the TKI group and 83.0% in the CO group (HR: 0.10; 95% CI: 0.01–0.78; Adjuvant EGFR‐TKIs could significantly improve DFS among patients with stage IB lung adenocarcinoma compared with CO, with a safe and tolerable profile. The inclusion criteria were as follows: patients who underwent complete pulmonary resection (lobectomy or sublobectomy) with or without lymph node dissection; life expectancy of at least 1 year; confirmed consolidation tumor ratio (CTR) >0.5 or pure solid nodule in chest computed tomography (CT); adenocarcinoma tumor pathology,2.2Adjuvant treatment is recommended for stage IB patients, especially those with one or more of the following high‐risk factors: poor cell differentiation, micropapillary component, solid component, visceral pleural invasion (VPI), sublobectomy, unknown lymph node status (pNx), lymphovascular invasion (LVI), and spread through air spaces (STAS). They chose to receive EGFR‐TKIs or observation according to their will and EGFR mutation status. Patients who underwent targeted treatment showed EGFR‐activating mutations in exons 19 or 21  in genetic tests on tumor tissue. They were recommended to initiate with the drug within 6 weeks after surgery and to take medication for more than 1 year. Three options were offered: 80 mg osimertinib once daily, 250 mg gefitinib once daily, or 125 mg icotinib thrice daily. Adjuvant‐targeted therapy can be discontinued until disease relapse or when unacceptable adverse effects occur. The study was performed in accordance with the guidelines for Good Clinical Practice and the principles of the Declaration of Helsinki, and was approved by the Ethics Committee of the Chinese People's Liberation Army General Hospital (S2021‐701‐01). All patients signed an informed consent form.2.3Routine examinations after surgery were requested every 3 months for the first 2 years, and every 6 months thereafter for 5 years. After 5 years, the patients were assessed annually. The examinations included blood tumor marker testing, chest CT, ultrasound of the neck and abdomen, and head magnetic resonance imaging. Bone scans were performed for patients with bone pain. Safety information, including clinical and laboratory adverse events, was also recorded for the EGFR‐TKI patients. Follow‐up information was collected through official contact with patients via telephone or from hospital outpatient clinic records. The last follow‐up was performed in August 2022. The primary endpoint was DFS, which was defined as the time from the surgery date to the first event  or the last follow‐up. DFS was calculated in months.2.4We conducted PSM to minimize the effects of observed confounders due to the imbalance of baseline characteristics between the TKI and clinical observation (CO) groups.2.5U test to determine significant differences in continuous variables between the two groups. Every group with categorical variables was summarized using the frequency and percentage of the considered population, and statistical comparisons between the two groups were performed using the chi‐squared test. DFS curves were estimated using the Kaplan–Meier method, and the log‐rank test was used to compare differences. The Cox proportional hazards model was applied in univariate and multivariate analyses to determine its influence on patient risk of recurrence. A two‐sided p < 0.05 was considered statistically significant.All statistical analyses were performed using SPSS version 22 (IBM Corporation). Continuous variables are expressed as means with standard deviation, as well as medians with a range of values. We used the Mann–Whitney 33.1p = 0.002), smoking history (p = 0.003), carcinoembryonic abnormality (p = 0.008), solid nodule (p = 0.006), EGFR mutation (p < 0.001), and cell differentiation (p = 0.018) before PSM. The two groups were comparable after PSM.Among the 555 patients diagnosed with stage IB disease after surgical resection between January 2013 and September 2021 at our thoracic center, 249 were recruited for this study  patients chose to receive icotinib. Sixteen patients 27.1%) received gefitinib, and five (8.5%) were treated with osimertinib. Forty‐three patients (72.9%) were administered TKIs for 2 years, and 16 (27.1%) received adjuvant treatment for 1 year (Table 7.1% rece3.3EGFR p.Thr790Met resistance mutation. One patient in the TKI group died of a cardiovascular event. Thirty‐seven patients in the CO group experienced recurrence or metastasis. Twenty‐eight (75.7%) patients received first‐line EGFR‐TKIs, and nine (24.3%) patients received chemotherapy with or without immunotherapy. One patient died of myocardial infarction, one of laryngocarcinoma, and 12 of lung cancer‐related causes in the CO group. Among the matched 118 patients, 2 (3.4%) patients in the TKI group and 10 (16.9%) patients in the CO group experienced disease relapse. No brain metastases were observed in the TKI group, whereas 2 patients experienced central nervous system metastasis in the CO group. The 2‐year DFS rates were 98.3% in the TKI group and 86.4% in the CO group . The 3‐year DFS rates were 98.3% in the TKI group and 83.0% in the CO group . Differences between the two groups were found in the entire cohort (p = 0.005)  . In the entire cohort, 15 patients died, and 39 patients had experienced recurrence or metastasis by the last follow‐up. Two patients in the adjuvant TKI group experienced recurrence or metastasis. One patient received chemotherapy plus immunotherapy, and another patient received osimertinib in the presence of the ) Figure , as well) Figure . The med3.4p = 0.048, p = 0.009, p = 0.044, p = 0.012, and p = 0.041, respectively) (Table p = 0.047), as well as poor cell differentiation , and STAS .Some variables, such as age, poor cell differentiation, micropapillary component, STAS, and targeted therapy, were significantly associated with DFS in the univariate analysis (y) Table . Additio4p = 0.008) compared with clinical observation, regardless of whether gefitinib, icotinib, or osimertinib were administered. None of the patients discontinued TKIs owing to the low occurrence rate of treatment‐related serious adverse events. However, the median DFS and OS data were immature owing to the relatively short follow‐up period.To the best of our knowledge, this real‐world study is the first to evaluate the efficacy and safety of adjuvant EGFR‐TKIs for stage IB lung adenocarcinoma based on the 8th edition AJCC/UICC stage classification, which demonstrated that adjuvant targeted therapy could significantly improve the 3‐year DFS rate .Precision medicine plays a leading role in lung cancer healthcare. With the groundbreaking progress made in targeted therapy for metastatic NSCLC, many clinical trials have focused on early stage and locally advanced NSCLC.Nevertheless, based on the inspring findings of the ADAURA trial, the NCCN guidelines recommend that high‐risk stage IB patients receive reasonable adjuvant chemotherapy or osimertinib, which other guidelines do not follow due to the lack of accumulated evidence. High‐risk factors include poorly differentiated tumors, LVI, wedge resection, VPI, and pNx.Previous randomized controlled trials (RCTs) have reported significantly longer DFS among patients who received adjuvant EGFR‐TKIs, compared with those who received chemotherapy. Two years of erlotinib treatment for stage IIIA NSCLC in the EVAN trial showed 81.4% 2‐year DFS, compared with 44.6% in the chemotherapy group.Regarding the safety of EGFR‐TKIs, the occurrence rate of adverse effects was consistent with that in previous trials.Our study had several limitations. First, the non‐RCT nature resulted in a slightly heterogeneous study population, and the number of enrolled patients was relatively low, both of which could be overcome through further RCTs with a large number of patients. Second, the difference between first‐generation (icotinib and gefitinib) and third‐generation (osimertinib) TKIs may influence survival results. Additionally, the different oral durations of TKIs may also be a confounder. Third, our study had a relatively short follow‐up time, which may not have provided sufficient OS information.5Adjuvant EGFR‐TKIs could significantly improve DFS among patients with stage IB lung adenocarcinoma, compared with CO, with a safe and tolerable profile.leilei shen: Conceptualization ; data curation ; methodology ; writing – original draft . Juntang Guo: Data curation ; writing – review and editing . Weidong Zhang: Formal analysis . Lianbin Zhang: Conceptualization ; supervision ; writing – review and editing . Xi Liu: Conceptualization ; supervision ; writing – review and editing . Tao Wang: Conceptualization ; supervision ; writing – review and editing . Tao Zhang: Conceptualization ; supervision ; writing – review and editing . Chaoyang Liang: Conceptualization ; supervision ; writing – review and editing . Yang Liu: Conceptualization ; project administration ; supervision ; writing – review and editing .This work was supported by the Special Science and Technology Projects for university and medical institutions of Sanya City (No. 2021GXYL44).The authors have no conflict of interest.The study was approved by the Ethics Committee of the Chinese People's Liberation Army General Hospital (S2021‐701‐01). All patients signed an informed consent form.


The current molecular classifications cannot completely explain the polarized malignant biological behavior of low‐grade gliomas (LGGs), especially for tumor recurrence. Therefore, we tried to identify suspicious hub genes related to tumor recurrence in LGGs.In this study, we constructed a gene‐miRNA‐lncRNA co‐expression network for LGGs by a weighted gene co‐expression network analysis (WGCNA). GDCRNATools and the WGCNA R package were mainly used in data analysis.Sequencing data from 502 LGG patients were analyzed in this study. Compared with recurrent glioma tissues, we identified 774 differentially expressed (DE) mRNAs, 49 DE miRNAs, and 129 DE lncRNAs in primary LGGs and ultimately determined that the expression of MKLN1 was related to tumor recurrence in LGG.This study identified the potential biomarkers for the pathogenesis and recurrence of LGGs and proposed that MKLN1 could be a potential therapeutic target. In this study, we constructed a gene‐miRNA‐lncRNA co‐expression network for low‐grade gliomas (LGGs) by a weighted gene coexpressionnetwork analysis (WGCNA). This study identified the potential biomarkers for the pathogenesis and recurrence of LGGsand proposed that MKLN1 could be a potential therapeutic target. We aimed to complement the characteristics of the lncRNA‐miRNA‐mRNA network in LGGs and ultimately identify multiple susceptibility loci and hub genes for LGGs.22.1GDCRNATools R package was used to obtain patient data from the National Institutes of Health (NIH)https://gdc.cancer.gov/) and to process these data. Briefly, GDCRNATools were used to process the Isoform Expression Quantification (BCGSC miRNA Profiling) data, Gene Expression Quantification (HTSeq‐Counts) data, and Clinical  data for the downstream analysis, such as differential gene expression analysis , Hypergeometric test, and Pearson correlation analysis. Finally, this R package can allow researchers to perform analyses by simply running a few functions and integrating their pipelines, such as molecular subtype classification and WGCNA.The 2.2The WGCNA R software package was used for finding clusters/modules of highly correlated genes.2.3https://gdc.cancer.gov/). A total of 502 LGG patients were enrolled in this study. All analyses were determined to be statistically significant, with p < 0.05. The plotted survival curves were constructed by survival and survminer R packages. In our study, we tried to use the receiver operating characteristic (ROC) curve to evaluate the best genes. The plotROC R package was used to generate the ROC curve.The molecular, clinical, survival, and histopathological data were acquired from the Genomic Data Commons database (GDC) (2.4starBase v3.0 (http://starbase.sysu.edu.cn/panCancer.php). starBase v3.0 is an open‐source platform for studying the miRNA‐mRNA, miRNA‐ncRNA, ncRNA–RNA, RBP‐mRNA, and RBP‐ncRNA interactions from the degradome‐seq, CLIP‐seq, and RNA–RNA interactome data. Gene co‐expression was found under the starBase Pan‐Cancer Analysis Platform.The criteria for correlation analysis are as follows: the expression values of genes from RNA‐seq data were scaled with log2 (FPKM +0.01), and the expression data of glioma were downloaded from the TCGA project via Genomic Data Commons Data Portal. The co‐expression correlation results were obtained from 2.5clusterProfiler R package was used to classify GO functions and to generate the GO and KEGG plots.To determine the biological mechanisms of the cohort of enrolled glioma patients, the differentially expressed genes (DEG) were subjected to GO and KEGG pathway analysis. KEGG enrichment analysis was imported into the KEGG database. The 33.1p < 0.05)   Figure , includi) Figure . The res3.2IntNMF R package to conduct nonnegative matrix factorization (NMF) cluster analysis, and the final cluster number k was set as K = 3  showed an obvious dismal prognosis compared with subtypes 1 (n = 134) and 2 (n = 230).To identify the potential DE lncRNAs, DE miRNAs, and DE mRNAs that could predict the overall survival (OS) period of LGGs, we used 3 Figure . In addi3 Figure , and the3 Figure . As demo3.3WGCNA R package to analyze the DE lncRNAs, DE miRNAs, and DE mRNAs among primary LGG and recurrent LGG . First, for each of the hub genes, the data were categorized into high and low groups using expression quartiles (n = 36/37 for each group). By using the ROC curve method, we identified the top three hub genes  with optimal sensitivity and specificity for discrimination between dead and alive patients. Kaplan–Meier log‐rank tests demonstrated that the high expression of MKLN1 and CYB5D1 indicated poor prognosis in the subtype Clust3 cohort but showed better survival for AC008764.2 than the counterparts .Subsequently, we analyzed the abovementioned 10 hub genes in the GDC subtype Clust3 cohort , we found that MKLN1 expression levels in all tumor groups were higher than those in normal counterparts  were identified. MKLN1 and CYB5D1 had an oncogene role and the high expression indicated poor prognosis in the subtype Clust3 cohort, however, AC008764.2 was associated with a good prognosis. Among them, MKLN1 holds the most optimal sensitivity and specificity in the AUC curve. MKLN1 (Muskelin 1) is an intracellular protein mediating cell spread and cytoskeletal responses to the extracellular matrix component thrombospondin I (MIM 188060).5In the present study, we constructed a ceRNA network and identified novel biomarkers, especially the MKLN1 gene, that have the potential for prognosis prediction in LGG patients. We hope that our research could provide a stratification marker for survival outcomes and could be a potential therapeutic target for LGGs. The biological mechanism by which dysfunction of the MKLN1 gene affects LGGs is expected in future research.Xiaodong Niu: Conceptualization (lead); data curation (lead); funding acquisition ; resources ; writing – original draft ; writing – review and editing . Qi Pan: Conceptualization ; funding acquisition ; resources ; software . Qianwen Zhang: Data curation ; resources . Xiang Wang: Supervision ; writing – review and editing . Yan‐Hui Liu: Conceptualization ; supervision ; writing – review and editing . Yu Li: Conceptualization ; supervision ; writing – review and editing . Yuekang Zhang: Conceptualization ; supervision ; writing – review and editing . Yuan Yang: Conceptualization ; funding acquisition ; software ; supervision ; writing – original draft ; writing – review and editing . Qing Mao: Conceptualization ; funding acquisition ; resources ; supervision ; writing – review and editing .This work was supported by the National Natural Science Foundation of China , the fellowship of China Postdoctoral Science Foundation (2022M712255), and the Spark Project of Sichuan University (No. 2082604401223).The authors have no conflict of interest to declare.All authors have read and approved the manuscript.Not applicable.


The cultural normativeness theory posits that specific parenting behaviors can be interpreted as displays of appropriate parenting in contexts where they are deemed normative. Previous studies suggest high acceptance of physical discipline in Singapore, where strict parenting could be interpreted as care for the child. However, there is a lack of studies on the local prevalence and implications of physical discipline. This study aimed to investigate the prevalence of Singaporean children experiencing parental physical discipline, longitudinal changes in this prevalence, and how exposure to physical discipline relates to children’s evaluation of their parents’ parenting.Participants were 710 children with parental reports of physical discipline at one or more assessments at ages 4.5, 6, 9, and 11 years in the Growing Up in Singapore Towards healthy Outcomes birth cohort study. Parental reports of physical discipline were obtained using the Parenting Styles and Dimensions Questionnaire or the Alabama Parenting Questionnaire across the four assessments. Child reports of their parents’ care and control were obtained using the Parental Bonding Instrument for Children at the age 9 assessment. Prevalence was specified as being exposed to at least one physical discipline at any frequency. A generalized linear mixed model was performed to examine whether children’s age predicted their exposure to physical discipline. Linear regression analyses were conducted to investigate whether children’s exposure to physical discipline predicted their evaluation of their parents’ parenting.SE = 0.01, OR = 0.87, p < 0.001). The more frequent the paternal physical discipline children were exposed to, the more likely they were to report lower levels of care  and higher levels of denial of psychological autonomy by fathers . Maternal physical discipline was not significantly associated with children’s evaluation of their mothers’ parenting (ps ≥ 0.53).The prevalence of children experiencing at least one physical discipline was above 80% at all ages. There was a decrease in this prevalence from age 4.5 to 11 years (B = − 0.14, Physical discipline was a common experience among our Singaporean sample, consistent with the notion that strict parenting could be regarded as a form of care. However, exposure to physical discipline did not translate to children reporting their parents as caring, with paternal physical discipline being negatively associated with children’s evaluations of paternal care.The online version contains supplementary material available at 10.1186/s13034-023-00632-9. Physical discipline refers to the use of physical force to inflict bodily pain, but not injury, with the intention of correcting or controlling the child’s behavior . Meta-anchiao shun) and ‘governing’ (guan) of the child through strict control tend to be associated with care and concern for the child in Chinese cultures, whereas such practices could be interpreted as forms of hostility in European-American cultures. Further, firm control and governance of the child could be considered as parental responsibilities in Chinese cultures, such that not engaging in such practices would be considered negligent and uncaring ). The effects of the interaction of children’s age with parent sex and physical coercion dimension item did not achieve significance (ps ≥ 0.21), suggesting that the decrease in the prevalence of physical discipline with age did not differ by parent sex or by the four items of the physical coercion dimension. We additionally conducted a linear mixed model with restricted maximum likelihood estimation entering the same fixed effects as in the model described above, but with children’s frequency of exposure to physical discipline  as the dependent variable. This model similarly yielded a significant effect of age, such that children’s frequency of exposure to physical discipline decreased with age  = 222.53, p < 0.001). The interaction between age and parent was nonsignificant  = 1.36, p = 0.24), but the interaction between age and physical coercion dimension item was significant  = 5.38, p = 0.001), such that the decline in frequency with age was largest for spanking and smallest for slapping. It should be noted that children’s age remained a significant predictor of children’s exposure to parental physical discipline even after controlling for children’s cognitive skills at age 4.5 years  = 1.83, B = − 0.55, SE = 0.41, nominal p = 0.18, adjusted p = 0.53), denial of psychological autonomy  = 0.19, B = 0.14, SE = 0.32, nominal and adjusted p = 0.66), and denial of behavioral freedom  = 1.72, B = 0.38, SE = 0.29, nominal p = 0.19, adjusted p = 0.53). In contrast, more frequent self-reported use of physical discipline by fathers was significantly associated with children reporting lower levels of care  = 6.92, B = − 1.74, SE = 0.66, nominal p = 0.01, adjusted p = 0.03) and higher levels of denial of psychological autonomy  = 5.59, B = 1.05, SE = 0.45, nominal p = 0.02, adjusted p = 0.04) by fathers. Fathers’ self-reported use of physical discipline was not significantly related to how children rated their fathers’ denial of behavioral freedom  = 0.40, B = 0.26, SE = 0.41, nominal and adjusted p = 0.53). While the analyses above examined concurrent relations between exposure to physical discipline and evaluation of parenting at age 9, supplementary analyses suggested that children’s exposure to maternal physical discipline at age 4.5 and 6 were also not significantly associated with how they evaluated their mothers’ parenting at age 9 , which includes respect and courtesy to parents, is rooted in Chinese culture, similar values may be shared by the Malay culture (ketaatan kepada ibu bapa) and Indian culture (seva) [The use of physical discipline is certainly not limited to the Chinese majority in Singapore, with our study finding that high prevalence rates were also shared by the Malay and Indian minorities. While the concept of filial piety (e (seva) . Prevalee (seva) , 39, 77.Our study was unable to capture a fine-grained picture of children’s physical discipline experiences. While many Singaporean children may have been physically disciplined by their parents, there can be wide variability in the delivery of physical discipline, including its frequency, severity, form, and impulsivity, as well as whether it is preceded by milder disciplinary techniques and accompanied by verbal reasoning. Circumstances that elicit parental physical discipline and the motivations behind parents' use of the practice could also differ across households, considering previous literature suggesting that parents' disciplinary choices  could differ depending on parents’ socialization goals  . Our stuPrevious research found that boys are more subject to physical discipline than girls, which could be due to factors such as gender expectations that boys require harsher discipline, or higher expectations for boys to be leaders within their household and the society in the future –37. HowePrevious studies, mostly conducted in Western countries, suggest that the prevalence of parental physical discipline tends to be high in early childhood and declines thereafter, potentially as children develop the sophisticated cognitive skills to reflect on and control their misbehavior, and mutual decision making becomes more prioritized in the parent–child relation –47, 83. Physical discipline could be interpreted by children as parental hostility or rejection, particularly in cultural contexts where physical discipline is non-normative , 12. A rThe impact of maternal physical discipline on children could be offset by the comparatively larger quantity of mother–child interactions that occur in a child’s daily life. In Singapore, mothers tend to be main caregivers to children and are more engaged than fathers in a wide variety of parenting practices that reflect involvement in their child’s life, warmth and acceptance towards the child, and guidance of the child . PreviouThis study suggests that the majority of children in Singapore experience at least one instance of parental physical discipline. What should follow is a careful examination of the implications of physical discipline, including the nature of its delivery, for child development given the Singaporean sociocultural context. The outcomes of a given discipline could depend on how children interpret the messages conveyed by the practice . One stuParents deserve extensive and compelling research on the potential outcomes of their disciplinary decisions, prior to being made recommendations about how their parental ‘toolbox’ techniques should be modified . It willAdditional file 1: Table S1. Contingency Table for Frequency at Which Children Were Physically Punished by Their Mothers as Reported by Their Mothers and Fathers. Table S2. Contingency Table for Frequency at Which Children Were Physically Punished by Their Fathers as Reported by Their Fathers and Mothers. Table S3. Contingency Table for Frequency at Which Children Were Spanked by Their Mothers as Reported by Their Mothers and Fathers. Table S4. Contingency Table for Frequency at Which Children Were Spanked by Their Fathers as Reported by Their Fathers and Mothers. Table S5. Contingency Table for Frequency at Which Children Were Slapped by Their Mothers as Reported by Their Mothers and Fathers. Table S6. Contingency Table for Frequency at Which Children Were Slapped by Their Fathers as Reported by Their Fathers and Mothers. Table S7. Contingency Table for Frequency at Which Children Were Grabbed by Their Mothers as Reported by Their Mothers and Fathers. Table S8. Contingency Table for Frequency at Which Children Were Grabbed by Their Fathers as Reported by Their Fathers and Mothers. Table S9. Prevalence of Physical Discipline of Children by Their Parents at Age 4.5, 6, 9, and 11 According to Child Sex. Table S10. Prevalence of Physical Discipline of Children by Their Mothers and Fathers at Age 9. Table S11. Prevalence of Physical Discipline of Children by Their Mothers at Age 4.5, 6, 9, and 11 by Maternal Ethnicity.