Ziziphus jujuba Mill. (jujube) is a well-known medicinal plant with pronounced wound healing properties. The present study aimed to establish the chemical composition of the lyophilized ethanolic extract from Romanian Ziziphus jujuba leaves and to evaluate the healing and anti-inflammatory properties of a newly developed lipophilic ointment containing 10% dried jujube leaves extract. The ultra-High-Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry method was used, and 47 compounds were detected, among them the novel epicatechin and caffeic acid. The extract contains significant amounts of rutin (29.836 mg/g), quercetin (15.180 mg/g) and chlorogenic acid (350.96 µg/g). The lipophilic ointment has a slightly tolerable pH, between 5.41–5.42, and proved to be non-toxic in acute dermal irritation tests on New Zealand albino rabbits and after repeated administration on Wistar rats. The ointment also has a healing activity comparable to Cicatrizin  on Wistar rats and a moderate anti-inflammatory action compared to the control group, but statistically insignificant compared to indomethacin in the rat-induced inflammation test by intraplantar administration of kaolin. The healing and anti-inflammatory properties of the tested ointment are due to phenolic acids and flavonoids content, less because of minor components as apocynin, scopoletin, and isofraxidin. Papaveraceae and Berberidaceae families, terpenes, saponins, and phloroglucinol derivatives (arzanol) [Research concerning healing wound therapy focuses on finding new herbal remedies that are assumed to have fewer side effects, lower cost and similar efficacy compared to conventional synthetic drugs ,2. Woundarzanol) ,10,11,12arzanol) ,15,16,17Plantago major L., Plantago lanceolata L. [Calendula officinalis L. [Aloe vera (L.) Burm.f. [Hypericum perforatum L. [Achillea millefolium L. [Matricaria chamomilla L. [Centella asiatica (L.) Urb. [Symphytum officinale L. [Helichrysum italicum (Roth) G. Don [Ziziphus jujuba Mill. (jujube), from the Rhamnaceae family. It is a native species of China, found today in temperate and subtropical climates. It is known for its nutritional (from its fruits) and medicinal value, several of its organs  being used in various ailments [Among the notable species used throughout the world for their wound healing action are olata L. , Calendunalis L. , Aloe ve Burm.f. , Hypericratum L. ,22, Achiolium L. , Matricamilla L. ,25, CentL.) Urb. , Symphytinale L. , and Hel) G. Don ,29. Anotailments ,31. Its ailments , for weiailments , and to ailments . It contailments ,36.The healing effects and the anti-inflammatory properties of the species’ leaves harvested from Romania have been attributed to phenolic acids and flavonoids, which are predominant, and the most active principles have been previously evaluated in two preclinical studies ,38. The Ziziphus jujuba Mill. leaves harvested from Romania. The study also aimed to evaluate in vivo the healing and anti-inflammatory properties of the dried ethanolic extract leaves after inclusion into a hydrophobic ointment base. For the selection of the ointment base, the aims were to choose safe, biocompatible, emollient, and inexpensive ingredients. Cholesterol is an important component of the extracellular lipophilic matrix of stratum corneum, one that ensures the skin barrier function [This study aimed to characterize chromatographically the phenolic and flavonoid content and other minor compounds of ethanolic dried extract obtained from function . It has function . It has function . Vaselinfunction . DiffereZiziphus jujuba Mill. leaves of indigenous plant harvested from Research Institute for Fruit Growing Pitesti, Romania was obtained according to the method indicated in a previously published paper [The dried ethanolic extract of ed paper . BrieflyHypericum perforatum (St. John’s wort), Calendula officinalis (marigold), Symphytum officinale (sorrel), Plantago lanceolata (plantain) and Chamomilla recutita (chamomile) containing as active principles phenolic acids, flavonoids and essential oil.Cetyl alcohol, cholesterol, refined coconut oil and petrolatum were provided by Fagron, Greece. Butylhydroxyanisole was purchased from Merck KGaA, Germany. Kaolin was purchased from Health Chemicals Co., Ltd., Zhangjiagang City, China and urethane from Sigma-Aldrich, Hamburg, Germany. Cicatrizin, produced by Pharmaceutical TIS, Bucharest, Romania, is an ointment that contains extracts of Indomethacin 40 mg/g ointment, used as a reference product in the anti-inflammatory assessment, is produced by Hyperion, Iași, Romania.w/w), Ziziphus jujuba Mill. leaves extract, cetyl alcohol, cholesterol and Vaseline were selected to form a single-phase basis suitable for active ingredient suspension. The coconut oil was chosen to adjust the ointment consistency and also for its antioxidant and natural fragrance properties [For the formulation of the lipophilic semi-solid pharmaceutical dosage form containing 10%  balance. The hydrophobic components  were melted together on a water bath heated at about 50 °C, and then the basis was cooled to 35 °C when butylhydroxyanisole was added and dissolved. The ointment base to be used alone as a control for the assessment tests, was prepared similarly but without extract inclusion.Organoleptic Properties and HomogeneityThe organoleptic characterization included appearance, consistency, and homogeneity, together with absence of phase separation, and instabilities of color ,51. GritThe pH measurements were determined as the European Pharmacopoeia recommendations by the potentiometric method . An inoLm/z range, at 70,000 resolution. Nitrogen was used as collision, sheath, and auxiliary gas at 11–48 arbitrary unit flow rates. The spray voltage was 2.5 kV, and the capillary temperature 320 °C. The energy of the collision-induced dissociation cell was varied in the 30–60 eV range. Calibrations were carried out in the 50–2000 μg/L concentration range, by serial dilution of the 10 mg/L methanolic standard mix. The lyophilized jujube leaves extract was dissolved in methanolic solution and filtered through a 0.45 μm polytetrafluoroethylene membrane before injection into the UHPLC-MS system. Quantitative data were evaluated by the Quan/Qual Browser Xcalibur 2.3 (Thermo Fisher Scientific). The mass tolerance window was set to 5 ppm for the two analysis modes. Individual phenolic acids and flavonoid contents were reported as μg/g lyophilized jujube leaves extract. Also, data processing, analysis, and interpretation using Compound Discoverer v. 2.1  software was performed using an untargeted metabolomics working template.Target phenolic acids and flavonoids analysis was performed with an UltiMate 3000 UHPLC System, coupled with a Q Exactive Focus Hybrid Quadrupole-Orbitrap mass spectrometer equipped with Heated Electrospray Ionisation (HESI) probe, all from Thermo Fisher Scientific, Bremen, Germany. Separations were performed on Kinetex  column (reverse-phase UHPLC column) and a gradient elution of a binary solvent system consisting of solvent A (water with 0.1% formic acid) and solvent B (methanol with 0.1% formic acid). Mass spectra were recorded in the negative ionization mode in the 100–1200 For materials in the semi-solid form, spreadability is an essential characteristic that reunites the rheological and structural properties. It is an important test in the assessment of topical semi-solid products, as it can accurately predict the behavior during dose disposal and application. The ointments’ spreadability mostly depends on the consistency and flowability of the base, but in the case of incorporating high amounts of active ingredients, they can influence the final performance.The spreadability was determined by using the extensiometric method, analyzing the deformation ability of the product when different weights were applied. The procedure was performed on both the base alone and on the pharmaceutical ointment, in triplicate. The device consists of two square plates of glass with 11 cm sides. The bottom plate is positioned over a millimetric graph paper on which five concentric circles are drawn. On the lower plate in the center of the first circle, 1 g of the sample was brought, then the second glass plate was placed. The diameter of the circle occupied by the ointment, after pressing with the glass plate weighing 145 g, was registered. At intervals of one minute, on the top plate of the extensiometer, weights of 50, 100, 200, and 500 g were gradually applied. The diameters of the circles formed by sample spreading were recorded each time ,55,56.2 and r is the radius in mm.The spreadability is calculated by the equation:The experiments were performed on animals purchased from the Cantacuzino Institute Biobase (Bucharest). The animals were acclimatized to laboratory conditions for five days before the start of the experiments. The room temperature during the treatment was 23 ± 1 °C, and the relative humidity was 50 ± 2%. The lighting was artificial, with a succession of 12 h of light and 12 h of darkness. The animals had unlimited access to conventional laboratory water and food .All the experiments complied with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes and the implementing Law no. 43/2014 on the protection of animals used for scientific purposes and were approved by the Bioethics Commission of the Faculty of Pharmacy, University of Medicine and Pharmacy Carol Davila, Bucharest (997/7 October 2016).Two tests were performed to determine the local tolerability of the newly formulated ointment, in accordance with the relevant OECD guidelines: acute irritation/corrosion and dermal irritation after repeated administration for 21 days. The OECD Guidelines for the Testing of Chemicals is a set of testing methods developed by experts from the OECD and used by various governments, companies, and independent laboratories to identify and characterize potential hazards of chemicals  and are To determine acute dermal irritation , the samA male New Zealand albino rabbit (4.55 ± 0.07 kg) was used. About 24 h before the test, the fur on the back was removed very carefully, so as not to damage the skin, using scissors and an electric razor.The studied ointment (ZIZ-L) (0.5 g) and lipophilic base (B-L) (0.5 g) were applied on a self-adhesive patch on a soft non-woven pad Cosmopore Advance 7.2 × 5 cm  and then affixed to the back of the rabbit, at a 5 cm distance from each other.After 4 h of application, the patch was detached, and the ointment was removed by gently wiping it with a cloth soaked in water.Following patch removal, the rabbit was evaluated for signs of erythema and/or edema immediately, and at 1, 24, 48 and 72 h. The observed dermal reactions were classified according to A determination of subchronic dermal toxicity  can be pZiziphus jujuba Mill. leaves is virtually nontoxic after single dose administration [If a dose of at least 1000 mg/kg bw of a sample does not produce detectable toxic effects, it is not necessary to use three levels of concentration. Previous research has shown that the dried ethanolic extract obtained from stration , and theThe sample is applied daily to the skin of the experimental animals (rats), in graduated doses, using several groups of laboratory animals, one dose for each group, for 21 days. During the application period, the animals are observed daily for signs of toxicity. Rats dying during the test are necropsied, and at the end of the test, surviving and uncropped animals are sacrificed.Group 1F: ZIZ-L-F: consisting of 4 females, who received the lipophilic ointment ZIZ-L;Group 1M: ZIZ-L-M: consisting of 4 males, who received the lipophilic ointment ZIZ-L;Group 2F: B-L-F: consisting of 4 females, who received the lipophilic base B-L;Group 2M: B-L-M: consisting of 4 males, who received the lipophilic base B-L.A community of 16 rats of 9 week old, Wistar strain, 8 females (214 ± 10 g) and 8 males (259 ± 13 g), were subjected to the tests. The animals were distributed into 4 groups, as follows:The fur was removed from the dorsal area of the torso 24 h before the test. This operation was repeated at intervals of about a week.Ointment and ointment base  were applied on a self-adhesive patch on a soft non-woven pad Cosmopore Advance 7.2 × 5 cm  and then affixed to the animal’s back to prevent it from gaining access to them. The animals were followed, the patch remaining set for at least 6 h after application. As OECD Guide 410 allows, ointments were applied 5 days/week for 21 days.Animals were monitored, in particular, for changes in the skin, fur, and mucous membranes, as well as in somatic-motor activity and for behavioral changes. The animals were weighed weekly.Male Wistar rats weighing 200 ± 10 g were used for the study. The rats were depilated in the dorsal area. After ethyl ether anesthesia, the animals suffered burn wounds using a metal device consisting of a disc with a 1 cm diameter which was heated in water with 5% NaCl at 105 °C. The heated disc was applied to the depilated dorsal area and held for 10 s ,61,62,63The rats were distributed by the randomization method in groups of 10 animals and were treated as follows:Group 1—control group, untreated;Group 2—group treated with lipophilic ointment ZIZ-L;Group 3—group treated with lipophilic base L-B;Group 4—group treated with Cicatrizin ointment, taken as a reference product .2) and comparing them with those of the untreated controls, respectively, with those of the treatment with Cicatrizin, the reference product.The treatment was given daily in a single application for 12 days. The evolution of the wounds was followed every two days by measuring the areas in the treated animals  were divided into three groups of 10 animals each, which were named according to the treatment received, as follows:The rats were anesthetized with a 13% urethane solution, administered intraperitoneally, in a dose of 130 mg/kg body weight. After the installation of general anesthesia, the initial volume of the right paw was determined.A quantity of 0.2 g of each ointment was applied to the surface of the right paw and massaged 50 times. Inflammation was generated by intraplantar administration of 0.2 mL kaolin 10% suspension, and the evolution of the induced edema was followed at 1, 2, 3 and 4 h.The evolution of paw edema was calculated using the following formula :The statistical analysis was carried out using GraphPad Prism v. 5.0.  and the computing and programming environment, R v. 4.2.0 .Results were expressed as mean ± standard deviation.t Student test was applied to compare two groups. One-way ANOVA and Tukey’s HSD were used to compare multiple groups. The statistical significance threshold was set at 0.05.Distribution normality was estimated using the D’Agostino & Pearson global test . The t SZiziphus jujuba Mill. leaves extract homogeneously suspended in the base, presented as fine particles, without the tendency to agglomerate or phase-separation. All these characteristics remained unchanged during the six months of preservation at room temperature.A greasy, unctuous, dark green ointment with a characteristic coconut and plant odor was obtained. It contains The pH of the lipophilic ointment was in the 5.41–5.42 range, while for the base the registered values were between 5.67 and 5.70. According to European Pharmacopoeia specifications, both semi-solid products have an easily tolerated pH, not being irritating when applied to the skin. After maintaining the products for six months at room temperature, a slight decrease in the pH values was remarked for both samples (5.36 for ointment and 5.62 for base).The analytical approach based on a target UHPLC-ESI/MS analysis allows the quantification of some bioactive compounds responsible for the bioactive potential of jujube leaves extract. The recorded chromatogram is presented in Ziziphus jujube Mill. leaves, similar to results being obtained by Xue (2021) [Quantitative analysis indicates that chlorogenic, 3,4-dihydroxybenzoic, and syringic acids were the primarily phenolic acids identified, while quercetin and rutin were the main flavonoids in the e (2021) . HoweverZiziphus jujuba Mill. leaves extract are quercetin [The lyophilized jujube leaf extract contains relevant amounts of rutin (29.836 mg/g) and quercetin (15.180 mg/g), and also chlorogenic acid (350.96 µg/g). The compounds which could be responsible for the anti-inflammatory activity of the ointment obtained with uercetin ,68, rutiuercetin , chloroguercetin , catechiuercetin , pinostruercetin , and feruercetin .The analytical approach based on non-target UHPLC-Q-Orbitrap HRMS analysis allows the identification of other bioactive compounds and specialized metabolites that occur in jujube leaf extract, which are also responsible for the anti-inflammatory activity. Phytochemical compounds such as flavonoids, organic acids, fatty acids, and other specific compounds such as apocynin, scopoletin, and isofraxidin show excellent anti-inflammatory activity ,75,76,77The compound’s name, retention time, exact mass, and accurate mass of m/z adduct ions in negative ESI mode for the identified compounds are shown in It is noted that the prepared ointment has proper plasticity, allowing it to easily spread on the skin . When reAreas exposed to the newly formulated ointment, as well as those exposed to the ointment base, were examined 4 h after application on rabbit, immediately after removal of the self-adhesive patches.In none of the cases was erythema or edema observed. The examination was repeated at 1, 24, 48, and 72 h after patch removal, and no signs of erythema or edema were detected .Ziziphus jujuba Mill. leaves is not irritating or corrosive following a single cutaneous application.After testing for acute dermal irritability according to OECD Guideline 404, it can be stated that the ointment obtained from For dermal irritation after repeated administration tests, the experimental results regarding the evolution of body weight during the 21 days can be found in The experimental results indicated no changes in the external appearance  or the motor performances of the rats in the two groups tested. Also, there were no alterations in somatic-motor activity or behavior.Ziziphus jujuba Mill. dried leaves extract.For all tested batches, the body weight increased  throughout the treatment, which indicates the lack of toxicity of both the base and the ointment containing the The results registered for the wound healing effect are shown in The experimental results on the wound healing are summarized in 2; after 12 days, it was sized 23 mm2, and showed a cure of 76.76% compared to the primary stage. Total healing occurred after 26 days.The control rats showed an initial burn area of 99 mm2. Complete recovery occurred after 18 days.The ointment used in the study as a reference product (Cicatrizin) resulted in 85.09% healing after 12 days of treatment and a burn area of 13.8 mmZiziphus jujuba Mill. ointment generated wound healing of 79.07% healing compared to the first day of treatment and a burn area of 20 mm2. Complete recovery occurred after 18 days.The lipophilic 2. Complete recovery occurred after 20 days.In the case of the animals treated with lipophilic base, 70.62% of healing was registered after 12 days and the burn area reached 28.2 mmThe experimental results on the evolution of edema induced by intraplantar injection of 0.2 mL 10% kaolin suspension are found in p < 0.001). At 4 h after administration of the inflammatory agent, the average increases in rat paws for the ZIZ-L and IND groups were similar .The administration of the inflammatory agent produced an increase in the volume of the rat paw for all three tested groups. As expected, the most pronounced increase in volume was observed in the case of rats from the control group, treated with B-L, 4 h after kaolin administration  at three hours and 15.78% (p < 0.05) at four hours, respectively.When compared to B-L activity, ZIZ-L ointment displays an anti-inflammatory effect, with a difference between average paw volume increases of 13.81% , a minor difference in the behavior was noticed after adding the extract to the base . The finZiziphus jujuba leaves was included in a high amount and was incorporated by suspension, leading to no deleterious dermal responses, including erythema and edema.Concerning the tolerance of the developed ZIZ-L ointment, the dermal irritation tests proved that it was well accepted, not inducing any reversible or non-reversible skin damage, and not affecting the somatic-motor functions. As animal skin is considered to be very sensitive to most pharmaceutical ingredients , the resZiziphus jujuba Mill. extract than those treated with the lipophilic base, suggesting that the extract owns a potent therapeutic effect in the wound healing process. The wound closure percentage displayed by the ZIZ-L ointment application was similar to the one achieved after the reference product administration, confirming that the proposed formulation has the potential for treating skin wounds. This supports the traditional use of this species leaves for wound healing, as reported in India. Whereas previous research reported wound healing activity for extracts prepared from this species’ fruits, bark or roots, our research was focused on a leaf extract prepared with 70% ethyl alcohol [This in vivo experiment confirmed that ZIZ-L ointment treatment significantly accelerated wound healing in Group 2, compared to control Groups 1 (untreated) and 3 (B-L treated). Wound healing resulted in a considerable decrease in the damaged epidermis length compared to controls. Visual examination showed that the epidermis recovery was faster in the wound treated with the ointment containing  alcohol ,85,86.Ziziphus jujuba Mill leaves extract enhances skin wound healing through multiple mechanisms. The major active ingredients that were identified in the extract were chlorogenic acid, quercetin and rutin. The mechanisms of action are complex. Chlorogenic acid promoted fibroblastic and remodeling phases of wound healing. It accelerated the wound closure in the presence of keratinocyte [tinocyte . Quercettinocyte . Also, itinocyte ,90. Rutitinocyte .Ziziphus ointment, the registered results show its potential properties. Even though the activity proved to be slower than the one displayed by the reference product, it was significantly higher than the lipophilic base action. The results support using the ointment as an herbal remedy for curative purposes in various topical inflammatory processes. Considering the chemical composition of Ziziphus jujuba Mill. leaves extract, its anti-inflammatory effect is due to its major constituents. Chlorogenic acid has antioxidant properties and because of that, it reduces the expression of inflammatory molecules. It inhibits phospholipase A2, cyclooxygenases and lipoxygenases, and reduces the concentrations of prostanoids and leukotrienes, especially PGE2 (Prostaglandin E2), IL-1β (Interleukin 1 beta), interferon-γ, monocyte chemotactic protein-1, and macrophage inflammatory protein-1α [Regarding the in vivo anti-inflammatory effect of otein-1α ,93. Querotein-1α ,95. Alsootein-1α ,97. It cotein-1α . Rutin iotein-1α . It alsootein-1α .Ziziphus ointment are mild and the epithelial tissue that are involved has an important regenerative potential [Ziziphus extract that have anti-inflammatory properties. This fact may avoid a determination of fibrosis [We have several reasons to consider that the wound healing process from this study consists of regeneration and not fibrosis. The injuries that we treated with the otential . The timfibrosis ,103.Ziziphus jujuba Mill. leaves’ ointment exhibited pronounced wound-healing effects and moderate anti-inflammatory characteristics, thereby supporting its usefulness as a medicinal therapy.Ziziphus jujuba Mill., formulated in a hydrophobic base consisting of petrolatum, but also cetyl alcohol, cholesterol, coconut oil and butylhydroxyanisole. The leaf extract used to prepare the ointment contains chiefly rutin (29.836 mg/g), quercetin (15.180 mg/g), and chlorogenic acid (350.96 µg/g); it also contains various amounts of phenolic acids, other flavonoids, fatty acids, organic acids and other compounds.The present evaluated an ointment containing 10% dried ethanolic extract from The formulated ointment has stable organoleptic properties related to those of the extract, is homogeneous, has a slightly acid pH (5.41–5.42), and appropriate rheological properties. It has demonstrated good tolerability following single and repeated administration in rat experiments. In rat wound models, the ointment resembled Cicatrizin in terms of its healing ability; both products accelerated healing, the effect being comparable.The tested ointment showed an anti-inflammatory effect compared to the control group. In comparison to indomethacin, although the effect was slightly more pronounced for the herbal ointment, the difference was not statistically significant.Due to its accessibility, good tolerance, and efficacy demonstrated in experimental models, the developed ointment is a promising therapy for wound healing and would be worth further exploring its benefits in a clinical setting.


To the Editor:Idiopathic pulmonary fibrosis (IPF) is a rare disease with prevalence of 50 in 100,000 cases in the UK.MUC5B gene promoter region (rs35705950) has a large effect on IPF risk with each copy of the T allele that is associated with a 4- to 5-fold increased risk of IPF.,Genome-wide association studies have identified 20 independent single nucleotide polymorphisms (SNPs) that are associated with IPF risk to date.Most datasets for genetic studies of IPF were derived from dedicated IPF cohort studies, registries, and clinical trials, which are usually modest in size. Large general population cohorts, such as UK Biobank, represent a valuable resource for increasing IPF case sample sizes for molecular epidemiologic studies. However, observed effect size estimates for rs35705950 on IPF risk in general population cohorts, for which cases are defined with the use of the International Classification of Diseases, revision 10 (ICD-10)Given this, we proposed that the IPF risk effect size of rs35705950 could be used to evaluate and refine the choice of codes to define IPF cases. We applied this approach in UK Biobank.UK Biobank is a prospective cohort study that contains > 500,000 volunteers who were recruited in the United Kingdom from 2006 to 2010 at ages 49 to 69 years.ICD-10 code J84.1  was used to define IPF from hospital episodes statistics (HES)  and death  data, which were available for all UK Biobank participants. Two self-reported pulmonary fibrosis variables were available. At baseline, participants were asked by a trained nurse to self-report any noncancer illnesses (field id 20002), which included “pulmonary fibrosis.” During an online follow-up survey about work environment conducted in 2015, 121,270 participants were asked whether a doctor had ever diagnosed them with IPF . Primary care data were available for 230,105 participants . Eight primary care codes (Read 2 and Read 3) were used to define IPF.Control subjects were defined as individuals who had linked primary care data that had not been defined as an IPF case in any of the data sources. We further selected control subjects to be similar to cases for age sex, ever-smoker status. Cases and control subjects were all of genetically determined European ancestry.Association of rs35705950 with IPF risk was tested with the use of logistic regression that was adjusted for the first ten genetic principal components. We compared the effect size (OR) of the association using each IPF definition with that reported by the largest genome-wide association study with the use of clinically defined IPF casesMUC5B SNP and compared the effect size to the gold standard. Specifically, we excluded (1) secondary or other causes of pulmonary fibrosis  defined dataset, we evaluated the effect of excluding participants with cooccurring ICD-10 codes (in HES or death) that might indicate misclassification. We then repeated the association by testing for the P < 5 × 10-8) for all but self-reported pulmonary fibrosis (P = 1.00 × 10-6)  A. For alRemoval of the cases with a cooccurring code that is suggestive of non-IPF pulmonary fibrosis or removal of the cases that are defined by the occurrence of a J84.1 code before January 2018 led to slightly closer effect estimates to those previously reported, but with substantially reduced sample sizes B.We used association of rs35705950 with IPF risk to evaluate code-based definitions of IPF in UK Biobank. We show that none of the available IPF code definitions, either individually or in combination, replicate the association effect size that is obtained with the use of clinically defined IPF cohorts. We observed that self-reported IPF in UK Biobank provided an effect estimate closest to those previously reported.We hypothesized that applying code-based exclusions to reduce misclassification among the cases would improve the effect estimates. Although this led to some increase in the effect sizes, they were still < 95% CI of the estimates from IPF studies that used tertiary care diagnoses to recruit participants. Excluding J84.1 ICD-10 code entries that occurred prior to January 2018 was more effective at increasing the OR on its own than removing cases with cooccurring medical conditions that can cause pulmonary fibrosis.The combined definitions of IPF in UK Biobank gave a prevalence of 559 of 100,000 cases, which is 10-fold higher than population estimates. Because IPF is a rare disease and we used a large control sample, the effect estimate attenuation that we observed for rs35705950 suggests that there is over-estimation of cases in UK Biobank because of low specificity of the definitions that are used.In conclusion, large biobanks offer an excellent resource for the study of less prevalent common diseases. However, we show that commonly used codes fail to define an IPF case sample that is able to replicate previously reported association effect sizes. Furthermore, pragmatic attempts to refine the phenotype with the use of further code exclusions were unable to improve the estimates. Researchers who use biobanks to study IPF should take these findings into consideration when designing future studies.L. V. W. holds a GSK/Asthma+Lung UK Chair in Respiratory Research (C17-1). L. V. W. and R. G. J. are supported by MRC Programme grant MR/V00235X/1. R. J. A. is an Action for Pulmonary Fibrosis Research Fellow. R. G. J. is supported by a National Institute for Health Research (NIHR) Research Professorship (NIHR reference RP-2017-08-ST2-014). L. M. K. was funded by a Medical Research Council (MRC) PhD studentship (MR/N013913/1). M. D. T. is supported by a Wellcome Trust Investigator Award (WT202849/Z/16/Z). A CC BY or equivalent license is applied to the Author Accepted Manuscript arising from this submission, in accordance with the grant’s open access conditions. The research was partially supported by the NIHR Leicester Biomedical Research Centre; This research has been conducted using the UK Biobank Resource under application 77050. This research used the SPECTRE High Performance Computing Facility at the University of Leicester.CHEST the following: L. V. W. reports current and recent research funding from GSK, Genentech, and Orion Pharma and consultancy for Galapagos. R. G. J. is a trustee of Action for Pulmonary Fibrosis and reports personal fees from Astra Zeneca, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Chiesi, Daewoong, Galapagos, Galecto, GlaxoSmithKline, Heptares, NuMedii, PatientMPower, Pliant, Promedior, Redx, Resolution Therapeutics, Roche, Veracyte, and Vicore. J. K. Q. has received grants from The Health Foundation, MRC, GSK, Bayer, BI, AUK-BLF, HDR UK, Chiesi. and AZ and personal fees for advisory board participation or speaking fees from GlaxoSmithKline, Boehringer Ingelheim, AstraZeneca, Chiesi, Insmed and Bayer. None declared .The authors have reported to


In this work we present a facile method for the fabrication of several capacitive transduction electrodes for sensing applications. To prepare the electrodes, line widths up to 300  The overall process leads to chemical wastage, poses environmental issues5 and customization in the electrode design is often expensive as the product cost relies heavily on the scale and batch size of fabrication. Therefore, contactless printing and contact printing, not requiring the provision of a clean room, have attained interest recently for R&D activities. Contact printing is widely used in the paper industry and print media. The upside of these printing strategies is their high throughput with accuracies up to 50 8. However, interconnect registration control, on account of tight tolerances and elastic nature of the substrate at high speed and pressure is intricate in nature. For large volume production the cost of printed features through roll-to-roll technology is cheaper than contactless printing method. However, for small production batches or customized imprints, the cost per item is a lot higher than contactless printing. Among contactless printing, inkjet printing has been widely used for printed electronic applications due to their low capital cost and pervasive availability. Moreover, compared to roll-to-roll printing technology the customized patterned printing can be done readily with the ability to print features or ink additively on the previously printed features. Thermal and piezoelectric inkjet techniques require formulation of ink, which needs to be compatible with the printing process. Ink often degrades in the thermal inkjet printing process if it is composed of material susceptible to thermal degradation, moreover high viscosity ink cannot be used with piezoelectric inkjet printers10. Screen printing for a simple lab-based R&D setup seems to be a possible solution for fabricating transduction electrodes at a much cheaper cost compared to the above-mentioned fabrication processes. Screen printing requires a stencil and although the process is simple, the low-cost customization of the transduction electrodes is a big issue and the process involves spreading a large amount of ink on the mesh. To circumvent the aforementioned issues, a simple laser ablation process for screen printing of conductive ink seems to a be an easier route for the fabrication of transduction electrodes. The laser ablation process from the commercial laser cutting machine not only provides facile implementation of transduction electrodes but also generates less ink waste when compared to the conventional screen printing of ink. In this work, printed capacitive structures to sense the electrochemical behaviour of the analyte are formed by laser ablation technique. The advantage of capacitive sensors is that they consume low energy, are less susceptible to radiation, have good sensitivity and provide fast response16. The most well-known design for measuring capacitive response is a parallel plate (PP) electrodes where the electrical terminals are isolated by a dielectric material18. For sensing applications and particularly in thin-film capacitive sensors, interdigitated electrodes (IDEs) are perhaps the most broadly utilized electrodes mostly due to their simple design, analytical and numerical modelling22.Numerous fabrication techniques have been reported in literature to form the transduction schemes for sensors attaining new functionalities, superior device responses and capabilities. However, most of the techniques require intricate processes and costly facilities to fabricate such sensors. For instance, the conventional microelectromechanical systems (MEMs) photolithography process, which is a top-down approach for fabricating sensing electrodes, requires cleanroom and chemical etching process23.The basic components of an electrochemical sensor are the sensing layer, transduction electrodes and the substrate. The sensing layer attracts the analyte by undergoing chemi-adsorption, which generates the electrical signal sensed by the readout circuit. The rate of adsorption dictates the response of the sensor where the desorption cycle is attributed to the recovery of the sensing layer. The sensing layer can be a single layer, bilayer or composite layer. A typical sensor layout is presented in Fig. 25. Other methods such as heating of substrate are also used to shorten or augment the recovery of such sensors26. Nevertheless, with proper selection of sensing layers, electrode geometry and suitable substrate a sensitive and highly responsive environmental sensor can be fabricated, which operates at room temperature with low or minimal sensor drift and without needing additional components28.Capacitive sensing has been commonly used for humidity sensors with reference capacitors so to mitigate the drift due to thermal interference. However, these devices are complex due to the inclusion of additional components29. In this scenario sensor fabrication techniques based on MEMs32, inkjet printing35 and contact printing38 methods can be expensive or unapproachable. However, a simple laser ablation technique by utilizing the desktop Fabricating the transduction schemes on the substrate requires intricate procedure and is often subjected to available resources. In the context of the prevailing COVID-19 pandemic situation most of the fabrication facilities are either non-accessible or closedA systematic methodology followed by pursuing the steps highlighted in Fig. mentclass2pt{minimThe microchannel is formed by the laser ablation process due to the absorption of energy induced by the laser beam. When the laser beam interacts with the workpiece it ablates the top surface of the workpiece. The rate of ablation depends upon the power, speed of the laser, wavelength of the radiation and material physical and optical properties. The resolution of the microchannel can be optimized by carefully selecting the parameters mentioned above. However, we have used the default ablation process parameters for ease of micropatterning and simplicity of fabricating micropattern, which can be utilized for sensing of humidity. In our experiments, we used 100% laser power and 100% speed for laser engraving on a 3 mm thick polymethyl methacrylate sheet. Figure Operations that were conducted to reduce the size of the suspended particles and to increase the activation sites can be seen from the scanning electron microscopy images in Fig. 40 and polyvinylpyrrolidone (PVP) coated silver nanoparticles42. Silver nanoparticles (SNPs) are known for their antimicrobial properties as in medical applications silver catheters and silver coated catheters are used for slow-injection of solvent while providing antiseptic properties. Moreover, as the nanoparticles are coated with PVP, there are less chances of toxicity and the possibility of oxidation45. On the other hand, cellulose is a good natural insulator commonly used as a dielectric material. It has been incorporated into many applications as substrate for conductive and non-conductive applications47.The sensing layer is based on biodegradable ink, which is mainly composed of cellulose, poly ethylene dioxythiophene: poly-styrene sulfonate (PEDOT:PSS)The sensing properties depend on the change in electrical properties of the sensing layer, which forms a uniform sensing region over the transduction electrodes. Once the sensing layer is exposed to humidity the porous structure of the cellulose decorated PEDOT:PSS and PVP coated SNP layer changes it’s capacitance during the adsorption and desorption cycles. The change in capacitance is then recorded for various humidity levels. All the sensors were tested with a starting relative humidity level of 50% as it was the prevailing ambient condition for conducting the humidity measurements. The measurements were taken with the help of GW INSTEK LCR-6000 Precision LCR Meter by sweeping the selectable frequencies between 100 Hz and 2 kHz as tabulated in Table The results of the humidity response with the transduction geometries are highlighted in Fig. The sensitivity of the sensor is defined as the ratio of difference of the capacitance at a specific relative humidity level designated by mentclass2pt{minimTable Figure The other reason is the gradual decrease of humidity level in the desorption cycle as compared to the adsorption cycle, which has a steep change in the humidity level. Since, the response of the DHT22 sensor has a higher rise due to the sudden increase of humidity in the chamber therefore, there is more hysteresis in the response stage of the sensor as compared to the recovery stage, where the hysteresis is low since the chamber humidity during this stage has a slower rate. It is anticipated that a precise measurement chamber can reduce the hysteresis between the response and recovery stage of the screen-printed sensors. It can be noted that in most of the cases the chamber starting humidity was a little higher at the end of the reading and the screen-printed response was also a little higher in the end depicting that there exists high correlation of humidity sensing of the sensors even with the slight deviation of humidity levels.In the preceding discussion the results of the sensitivity, hysteresis, response and recovery of various transduction scheme was presented for humidity sensing. It is pertinent to note here that the sensitivity of the meander electrode was higher among all the fabricated sensors however, the response and recovery cycle was the lowest among all the geometries. Moreover, there was an appreciable increase in the capacitance value above 80% relative humidity, which indicates highly non-linear relationship between the humidity and respective capacitive response. In this regard, the most promising electrode configuration seems to be either serpentine and custom pattern as these were the only geometries, which provided a good sensitivities and less non-linearity among all the fabricated geometry.Laser scribing is a method to induce high laser power to produce features or cuts on the surface of the substrate. In a mechanical workshop, the laser machine is used for cutting various materials of different thickness to perform 2D cutting and engraving. The main purpose is to transform the digital design to follow a laser path, which can be used for either cutting or engraving purposes. The depth of the cut depends upon the settings of power of the laser, speed of the laser and whether the spot size of the laser is focused on the substrate. On the other hand, the width of the cut depends upon the focusing lens, laser spot speed, power of the laser and distance of the laser with the object. If the laser is adjusted so as to focus properly on the substrate, then the quality of the laser cut is precise and is slightly above the focused spot size of the laser. The power of the laser is converted into heat energy when focused on the substrate and removes the material by locally ablate or burn the material to induce the digital imprints on the substrate. In the laser cutting process, the cut width is often termed as the kerf width of the laser cutting process. In order to reap the benefits of the above-mentioned process, we followed a facile process of engraving the designed pattern on the Poly (methyl methacrylate) (PMMA) sheets of thickness 3 mm. The printing process involves the computer-aided (CAD) designs of the electrode geometry and digitally transforming those through The laser ablation was done by considering the glass transition temperature of the polymer substrate, laser speed, laser power and z-height of the laser beam without the optimization of the kerf width. This technique provides a rapid production of custom geometrical transduction scheme on the substrate. Due to the nature of the process, it requires no special micro fabrication process and can be done in a workshop environment to produce the transduction electrodes. Moreover, as the polymer becomes flexible close to its glass transition temperature, there is a possibility to adhere the substrate to curved surfaces. Moreover, the geometrical designs can be readily exported to the laser cutter, where the laser can be turned on and off instantly and the engraving/cutting features can be set for various layers. However, the limitation of the above technique is the resolution of the laser beam and the accuracy of stages of the Once an ample amount of HPS-021LV was coated on the PMMA substrate the ink settles insides the engraved geometrical features. Later these geometrical features were subjected to heating in a convective oven to evaporate the solvent at 100 The process of ink preparation involves the synthesis of ultrafine particles from an amorphous precursor. For this purpose, a comprehensive methodology has been devised. The methodology involves the following steps as shown in Fig. The process starts with the wet grinding of 1 g of Sigmacell Cellulose (Product Code: S3504) of Type 20 with 20 48. Once the mixture was prepared the solution was poured on the transduction electrode and each transduction acrylic plate was then spin-coated at 1000 rpm for 120 s for each type of transduction geometry.Later the mixture was transferred into the beaker and was weighed to record the concentration of solvent in the mixture. Once the weight measurement was taken, 50 ml of deionized water was added so facilitate the stirring process through a magnetic stirrer. Magnetic stirring of mixture provided a uniform homogenization of amorphous solid particles in the solvent. Thus, improving the uniformity of the suspended particles in the solution. After the stirring process the mixture was heated to 100 49. To test the performance of various patterns of electrodes, we chose to work with the most frequently measured physical quantity i.e. humidity. This provided us with a base for assessing performance parameters of various patterns of electrodes, built using in-house facilities. Since, the humidity level in an indoor setting is quite low and stable, we carried out the experiments in an environment where humidity could be controlled to monitor the behaviour of the electrodes. Therefore, an environment was built in a plastic container that was linked to an external humidifier where the level of humidity was varied and continuously monitored. The basic layout of the experimental setup is depicted in Fig. Transduction electrodes are commonly used in sensing applications. The electrodes provide the ability to measure different kinds of gases such as Nitrous oxide, gaseous Ammonia, humidity, and many moreThe container was built such that the DHT22 sensors and different patterns of electrodes could be placed inside it. To achieve homogeneity of humidity inside the container, two acrylic plates were placed horizontally inside the container with holes made using a laser cutter. The holes of 5 mm in diameter were spread evenly across both plates with 15 mm spacing between them, and a 7 mm offset in holes between the top and bottom plate. The bottom plate assisted in dispersing humidity evenly while the top plate assisted in releasing excess humidity out of the chamber. Four DHT22 humidity sensors were placed on all four sides of the container such that the sensors and the electrode sensors were placed vertically around the inner walls of the container located between the top and the bottom plates. This enabled us to simultaneously test the performance of all the different patterns of transduction electrode sensors with varying levels of humidity. The two plates with holes fit inside the container with a much larger hole cut out at the centre of the container for a plastic pipe that drops down to the humidifier.Humidity can easily be generated using an appliance called the humidifier, which is inexpensive and provides the user with the ability to control the humidity. The humidifier used in the experiment is a droplet free ultrasonic cool mist generator  and has a 1.5 l water tank (Kogan Mini 1.5L Humidifier). The humidifier was placed at the bottom of the container with a circular hole cut out at the bottom for a tight fit of a 30 mm wide pipe connecting to the outlet of the humidifier. A support structure was built to place the container on top of the humidifier. The humidifier exudes mist from the top into the bottom of the container through a pipe. Each transduction pattern has two square pads filled with silver ink that extends to the sensing electrodes themselves. With the help of adhesive copper tape, small pieces were fixed to the two pads, so that the thin multistrand wires could be soldered on to the tape for connectivity. As soon as the humidifier starts introducing humidity into the container, DHT22 sensors begin reading humidity levels and simultaneously the transduction electrodes start sensing the humidity levels. Humidity levels from DHT22 sensors, were read using Arduino Nano that was kept separate from the capacitive readings taken with Nucleo-F446RE board. Components of the experimental setup are labelled in Fig. The top view of the experimental setup indicates the position of the transduction electrodes of six different patterns  and four DHT22 humidity sensors, all mounted on inner walls of the container.In this work, we presented a comparison among six different electrode layouts fabricated by using a laser ablation process. It has been observed that depending on the specific application and its requirements, an appropriate transduction scheme for environmental humidity sensors can be ascertained. For a large area sensing applications, the presented designs are scalable and suitable for sensing applications. The custom triangular pattern presented in this work can be a promising scheme when scalability for large area is not an issue. The fabricated sensors were tested at various relative humidity levels that achieved a good sensing response with sensitivities ranging from 0.13 to 2.37 pF/%RH in general for various transduction schemes. The meander geometric transduction scheme reported the highest sensitivity among the fabricated sensors however, there were some demerits for this geometry, such as lower response and recovery time along with associated non-linearity of capacitive response with respect to humidity. The work presented here provides a facile approach, biocompatible sensing layer and compendium of processes for fabricating sensors in a small low-cost laboratory, which can be of great advantage during the prevailing COVID-19 pandemic. Furthermore, the results obtained from the presented fabrication scheme can be extended for a high-resolution patterning electrode geometry with a suitable sensing layer.


Edwardsiella piscicida. However, the regulation of secretion by EscE is incompletely understood. In this work, we reveal that EscE interacts with EsaH and EsaG. The crystal structures of the EscE-EsaH complex and EscE-EsaG-EsaH complex were resolved at resolutions of 1.4 Å and 1.8 Å, respectively. EscE and EsaH form a hydrophobic groove to engulf the C-terminal region of EsaG (56 to 73 amino acids [aa]), serving as the cochaperones of T3SS needle protein EsaG in E. piscicida. V61, K62, M64, and M65 of EsaG play a pivotal role in maintaining the conformation of the ternary complex of EscE-EsaG-EsaH, thereby maintaining the stability of EsaG. An in vivo experiment revealed that EscE and EsaH stabilize each other, and both of them stabilize EsaG. Meanwhile, either EscE or EsaH can be secreted through the T3SS. The secondary structure of EsaH lacks the fourth and fifth α helices presented in its homologs PscG, YscG, and AscG. Insertion of the α4 and α5 helices of PscG or swapping the N-terminal 25 aa of PscG with those of EsaH starkly decreases the protein level of the chimeric EsaH, resulting in instability of EsaG and deactivation of the T3SS. To the best of our knowledge, these data represent the first reported structure of the T3SS needle complex of pathogens from Enterobacteriaceae and the first evidence for the secretion of T3SS needle chaperones.The intracellular EscE protein tightly controls the secretion of the type III secretion system (T3SS) middle and late substrates in  The type III secretion system (T3SS) is a protein secretion machinery that delivers virulence effectors from the cytosol of a variety of Gram-negative pathogens directly into the host cell cytoplasm. The injectisome is composed of membrane-embedded protein rings extended by a hollow needle formed by a single polymerized shaft, through which effectors are secreted or translocated to aid infection.Edwardsiella piscicida, previously known as Edwardsiella tarda , Shigella, Salmonella pathogenicity island 2 (SPI2), Salmonella SPI1, Burkholderia, or Chlamydia trachomatis is composed of a single protein: YscF, PscF, AscF, EscF, MxiH, SsaG, PrgI, BsaL, or CdsF, respectively  shares sequence homology with SsaE in Salmonella enterica, YscE in Yersinia enterocolitica, and CV2595 in Chromobacterium violaceum  and InterPro (http://www.ebi.ac.uk/interpro/search/sequence) for informatic analysis, EsaG was found to belong to the T3SS_needle_F superfamily and shares homology with the type III needle protein MxiH of Shigella, YscF of Yersinia pestis, SsaG of Salmonella enterica, PscF of P. aeruginosa, and EscF of EPEC. Notably, EsaG shares 61.8% and 64.4% similarity with SsaG and EscF, respectively, and 42.6%, 43.0%, and 45.3% similarity with YscF, AscF, and PscF, respectively  EscG, 25% similarity with Y. enterocolitica YscG, 16.8% similarity with A. hydrophila AscG, and 7.7% similarity with P. aeruginosa PscG (The iscicida . The esaon assay . EsaH shosa PscG .escE gene (previously known as orf13) is located upstream of esaG, spaced by esrC in the T3SS gene cluster of E. piscicida , EscE is soluble and stable, while EsaH is insoluble and unstable. By coexpression and copurification, we obtained the EscE-EsaH complex. The crystal structure of the EscE-EsaH complex was solved by single-wavelength anomalous diffraction (SAD) at a resolution of 1.8 Å, and it has been refined to an Rwork/Rfree of 20%/22.8% with good geometry  mediate the interactions between EsaH and EscE  and ΔesaG, ΔescE, and ΔesaH mutant strains were compared. It was observed that disruption of EsaH, EscE, or EsaG obstructed the secretion of the translocon proteins EseB, EseC, and EseD and effector protein EseJ, as revealed by SDS-PAGE and Coomassie blue staining. Complementation of the ΔesaG or ΔesaH strain restored their secretion profile to that of the WT strain  only in the cell lysates of the WT strain, but not in those from the ΔesaH, ΔescE, or ΔesaG strain . Therefore, the posttranslational links among them might be involved.But how do these three proteins influence each other? To investigate, the intracellular steady-state protein level of EsaG, EsaH, or EscE was examined in the WT and ΔG strain . This inesaG, obtaining WT 3×FLAG::esaG, ΔescE 3×FLAG::esaG, and ΔesaH 3×FLAG::esaG strains. The three strains were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with chloramphenicol (Cm) to inhibit protein synthesis, and the steady-state protein levels of EscE, EsaH, and EsaG were examined at 60-min intervals until 360 min after chloramphenicol treatment. As shown in escE strain sharply decreased, as did the steady-state protein levels of EscE and EsaG in the ΔesaH strain. These results indicate that both EscE and EsaH are essential for maintaining the stability of EsaG, while EscE and EsaH are mutually required for their stability.The stabilities of EsaG, EsaH, and EscE in the absence of one another were determined. Given the low efficiency of the EsaG antibody in probing the intracellular EsaG, we incorporated a 3×FLAG tag at the N terminus of chromosomal Rwork/Rfree of 20.4%/23.2% with good geometry  were measured as the readout of the translocation of EsaH::CyaA, EscE::CyaA, or EseG::CyaA. As shown in esaN mutant-infected cells were similar to that of the uninfected control for EsaH::CyaA. The cAMP levels in WT-infected cells were 202.99 ± 63.57 fmol/μg of host protein for EseG::CyaA (positive control) and 76.89 ± 14.91 fmol/μg of host protein for EscE::CyaA (positive control) when the cAMP level of EseG::CyaA or EscE::CyaA was less than 6.0 fmol/μg of host protein in cells infected with the ΔesaN strain (negative controls). These results indicate that, different from EscE, which is translocated into host cells in a T3SS-dependent manner, EsaH is not translocated into J774A.1 cells. Next, we investigated whether the failure in EsaH::CyaA translocation is due to its incapability of being secreted. To investigate, extracellular proteins (ECPs) of the WT/pACYC-esaH::cyaA or ΔesaN/pACYC-esaH::cyaA strain were harvested before being probed with anti-EsaH and anti-EvpC antibodies. EvpC was used as a loading control. It was found that EsaH-CyaA was secreted from the WT but not ΔesaN background . Moreover, as the secretion signal of T3SS substrates is always located in their N-terminal regions, 1 to 25 aa of EsaH were swapped with 1 to 25 aa of PscG, obtaining pJN-pscG1–25 aa-esaH26–88 aa-HA (pJN-esaHmut2-HA). pJN-esaH-HA, pJN-esaHmut1-HA, and pJN-esaHmut2-HA were introduced into the ΔesaH strain, and their expression was induced with l-arabinose. It was observed that insertion of the α4 and α5 helices of PscG into EsaH moderately decreased the steady-state protein level of EscE, while swapping the N-terminal 25 aa with that of PscG starkly decreased EscE, meanwhile, the intracellular EsaG was undetectable under both conditions. The steady-state protein level of EseG, EseB, or EvpC in each strain did not change with the expression of the chimeric EsaH , while no EsaH can be detected, emphasizing the fact that EscE is stabilizing EsaH. Moreover, in this study, we demonstrated that EsaH is the main chaperone that binds and stabilizes EsaG. Five helices of EsaH create a concave surface and a highly hydrophobic platform that “grasps” the C-terminal helix of EsaG56–73. The EscE-EsaH complex maintains the carboxyl terminus of the proteins’ cognate fiber-forming partner EsaG in a coiled-coil interaction, thereby stabilizing EsaG and ensuring its proper secretion. Secretion of EsaG leads to the secretion of T3SS middle and late substrates. In this study, we revealed that EscE regulates the secretion of T3SS substrates through controlling the steady-state protein level of EsaG, whose secretion initiates the secretion of T3SS middle and late substrates.Secretion of the T3SS translocon or effectors is obstructed when the T3SS needle chaperones are disrupted in EPEC, himurium , 36, 37.himurium , 38, 39.lla SPI2 . In E. pE. piscicida and abolishes the secretion of EsaG or EscE. It is speculated that inserting the α4 and α5 helices into EsaH may destroy the interaction between EsaH and EscE or EsaH and EsaG, thereby impeding the secretion of T3SS middle and late substrates. Moreover, the RMSDs of EsaH/PscG and EsaH/YscG were 3.7 Å and 3.1 Å, respectively, indicating that, unlike PscG and YscG, EsaH may be a novel chaperone. The tetratricopeptide repeat region (TPR) is a structural motif of T3SS needle chaperones, and EsaH may form a partial TPR fold, which enables its secretion.Seven helices were detected from PscG, YscG, and AscG by secondary structure analysis, and five from EsaH and SsaH, which lacked the fourth and fifth α helices of their homologs. EsaH can be secreted through the T3SS. Inserting the α4 and α5 helices of PscG between the third and fourth α helices of EsaH decreases the steady-state protein level of the chimeric EsaH inside E. piscicida can be translocated into host cells, where it interacts directly with mammalian factor Cugbp2 . Hence, we speculate that EsaK might interact with the EscE-EsaG-EsaH complex and contribute to the secretion of EsaG. Moreover, EsaK shares 19% similarity with SsaK . SsaK isE. piscicida.Type III secretion occurs in a hierarchical manner : proteinSalmonella stimulates gastroenteritis in humans and typhoid-like fever in mice. Salmonella SPI2 is switched on in response to the acidic pH inside the Salmonella-containing vacuole and is required for the establishment of systemic infection  (BD Biosciences) or in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) under a 5% (vol/vol) CO2 atmosphere to activate T3SS. When required, appropriate antibiotics were supplemented at the concentrations of 12.5 μg/mL colistin (Col) (Sigma), 34 μg/mL chloramphenicol (Cm) (Amresco), 15 μg/mL tetracycline (Tet) (Amresco), and 50 μg/mL gentamicin (Gm) (Amresco).The PD130/91 . The E. esaH or esaG gene was deleted from the chromosome of E. piscicida PPD130/91 by sacB-based allelic exchange using a method previously described (-esaH-HA and pJN-esaG-HA are derivatives of pJN105 (esaH or esaG gene under the control of l-arabinose-inducible araBAD promoter. The fourth and fifth α-helix sequences of the pscG were inserted into esaH to obtain pJN-esaHα1–3-pscGα4–5-esaHα4–5-HA (pJN-esaHmut1-HA). In a similar way, pJN-pscG1–25 aa-esaH26–88 aa-HA (pJN-esaHmut2-HA) was constructed. The plasmids acquired were verified by DNA sequencing before being transferred into E. piscicida strains and their expression was verified by immunoblotting.The escribed , 50. Thef pJN105 , which cE. piscicida extracellular proteins (ECPs) and bacterial lysates  were prepared as described by Zheng and Leung (E. coli BL21(DE3), the EsaG antibody was raised in rabbit against aa 18 to 31 (SNDAHSVITSGNVN), which was conjugated with keyhole limpet hemocyanin, and the antisera obtained were purified using the specific peptide as the ligand . To probe EscE, EsaH, or EsaG, proteins transferred onto PVDF membrane (0.22-μm pore) were fixed in 2.5% glutaraldehyde for 1 h before being subjected to blocking and immunoblotting.nd Leung . They wend Leung . For immnd Leung , EvpC  and a GAL4 activation domain-encoding prey vector (pGADT7) in the yeast strain AH109, as described by Liu et al. . All fusescE gene was cloned into a glutathione S-transferase (GST) fusion protein expression vector pGEX-6P-1  and esaH into pET-28b. The two plasmids obtained were cotransformed into E. coli BL21(DE3), and cultures were grown at 37°C to an optical density at 600 nm (OD600) of 0.6 to ~0.8, before induction with 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG)  for 16 h at 20°C. The harvested cells were sonicated in buffer A . Cell lysate was centrifuged at 18,000 × g for 1 h at 4°C to collect supernatant, which was incubated with glutathione Sepharose 4 FastFlow beads  at 4°C for 3 h. The proteins on beads were digested with 3C protease. The protein complex was further purified by size exclusion chromatography (Hiload 16/600 Superdex 75 prep grade) in buffer B  as the running buffer. Purified protein was analyzed by SDS-PAGE. The peak fraction was pooled and concentrated to 15 mg/mL. Selenomethionine-labeled EsaH/EscE was purified the same way as the unlabeled proteins.The 6-esaG33–73-esaH and pET-28b-escE were cotransformed into E. coli BL21(DE3), and the three proteins were purified using Ni-nitrilotriacetic acid (NTA) beads . The proteins were eluted from the beads with Ni-binding buffer containing 0.25 M imidazole, 400 mM NaCl, and 50 mM Tris-HCl (pH 8.0). Proteins were further purified on HiLoad 16/600 Superdex 75 prep-grade  gel filtration column in buffer B. The proteins purified were analyzed by SDS-PAGE. The peak fraction was pooled and concentrated to 19 mg/mL.To copurify the three proteins, pETDuet-1-His33–73-EsaH complex crystal was developed with 3.3 M NaCl and 0.1 M each KH2PO4 and Na2HPO4 (pH 6.2) at 16°C. All the diffraction data were collected at Shanghai Synchrotron Research Facility (SSRF) on beamline BL17U, BL18U, or BL19U, integrated, and processed with the HKL2000 program suite and XDS packages , 23% PEG monomethyl ether 2000, supplemented with a final concentration of 3% (wt/vol) 1,6-hexanediol, and were developed for 2 days at 16°C. The EscE-EsaGpackages . The strm Molrep . Refmac5m Molrep  was usedhttp://www.pymol.org/).All of the structures were iteratively built with COOT  and refiesaG strain, respectively. In parallel, the wild-type copy of esaG was inserted into pJN105, and the resulting plasmid was introduced into the ΔesaG strain, which was used as the control for the EsaG stability assay. The steady-state protein levels of EsaG obtained from the strains described above were probed.According to the structure information on the interaction between EsaG and EsaH/EscE, EsaG V61A, K62A, M64A, M65A, I68A, I69A, I72A, V61A K62A, M64A M65A, I68A I69A, M64A M65A I68A I69A, and K62A M64A M65A I68A I69A site mutations were obtained through gene synthesis , and the DNA fragments were ligated into pJN105. The plasmids obtained were introduced into the ΔThe CyaA translocation assay was conducted on J774A.1 macrophage cells, as previously described , and cycE. piscicida WT 3×FLAG::esaG, ΔescE 3×FLAG::esaG, and ΔesaH 3×FLAG::esaG strains were constructed as reported previously , anti-EscE, anti-EsaH, or anti-DnaK antibody. DnaK, a chaperone protein, was used as a loading control. The protein stability experiment was repeated at least three times, and representative images are shown.The eviously , subcultt test. Results are expressed as the mean ± standard deviation (SD).The results were analyzed using GraphPad Prism 6. Statistical significance was calculated using the two-tailed Student's E. piscicida PPD130/91 EscE-EsaH and EscE-EsaG33–73-EsaH have been deposited in the Protein Data Bank under PDB ID 7Y6B and 7Y6C.The atomic coordinates of


The causal relationship between HPV and cervical cancer in association with the high prevalence of high risk HPV genotypes led to the design of HPV vaccines based on the major capsid L1 protein. In recent years, capsid protein L2 has also become a focal point in the field of vaccine research. The present review focuses on the variability of HPV16 L1 and L2 genes, emphasizing the distribution of specific amino acid changes in the epitopes of capsid proteins. Moreover, a substantial bioinformatics analysis was conducted to describe the worldwide distribution of amino acid substitutions throughout HPV16 L1, L2 proteins. Five amino acid changes , ,  are frequently observed in the L1 hypervariable surface loops, while two amino acid substitutions  are adjacent to L2 specific epitopes. These changes have a high prevalence in certain geographic regions. The present review suggests that the extensive analysis of the amino acid substitutions in the HPV16 L1 immunodominant loops may provide insights concerning the ability of the virus in evading host immune response in certain populations. The genetic variability of the HPV16 L1 and L2 epitopes should be extensively analyzed in a given population. Human Papillomaviruses (HPVs) are non-enveloped, double–stranded circular DNA viruses of 8 kb in size that infect basal keratinocytes of mucosal and cutaneous epithelia ,2. BasedHPVs infect the basal layer of the epithelium via micro-wounds ,13. To iHPVs have established molecular mechanisms in order to prevent cell-cycle arrest and apoptosis. The viral E6 and E7 oncoproteins are involved in the uncontrolled cell proliferation as they inactivate tumor suppressor proteins p53 and pRB, respectively ,23,24. TThe genome of HPV16 is organized in three major regions; the long control region (LCR), the early region that encodes for the early genes E1, E2, E4, E5, E6 and E7, and the late region that encodes for the late genes L1 and L2 ,28,34. ACervical cancer is the fourth most common type of cancer and the second leading cause of cancer-related death in women in developing countries . CytologIn the present study, we review the non-synonymous nucleotide variations that have been reported in the L1 and L2 capsid genes of HPV16 DNA with a special focus on those which are located in antigenic epitopes. Moreover, the impact of the L2 minor protein on prevention of viral transmission is also discussed. Finally, we performed a large-scale analysis of publicly available HPV16 L1 and L2 gene sequences in order to identify the global distribution of particular amino acid changes which may affect the aggression of the virus.Currently, the US Food and Drug Administration (FDA) has approved three prophylactic HPV vaccines. In 2006, the FDA first approved Gardasil (Merck), which provides protection against four HPV genotypes . Next, Cervarix (GlaxoSmithKline) was approved by the FDA in 2009, and it provides immunity against HPV16 and HPV18. Finally, in 2014 the FDA approved a supplemental use of Gardasil 9  expanding its administration in women and men of 27 to 45 years of age. Gardasil 9 protects against HPV 6, 11, 16, 18, 31, 33, 45, 52 and 58 . The des+T cells. CD4+T cells in turn secrete cytokines, thus leading to the activation of B-cells, T-cells and macrophages [>The HPV VLPs have a highly ordered structure that exposes the L1 epitopes, promoting T-cell activation. In particular, the major histocompatibility complex class II molecules (MHC-II) that are expressed in the antigen-presenting cells (APCs) enable the presentation of the L1 epitopes to CD4rophages ,57,58,59rophages ,61. Notarophages   ,76. ConsThe analysis of the L1 gene revealed that non-synonymous mutations were situated adjacent to the neutralizing epitopes, thus offering capsid L1 protein a considerable advantage to avoid immune response . In partp < 0.002) increase in antibody titer, while T379P presents a 0.4 fold decrease in antibody titer [Research data collected from different studies revealed that numerous amino acid changes are found throughout the L1 ORF, while a considerable variability is detected near or within the region of the L1 gene that encodes for the respective immunodominant loops. In particular, a total of twenty-seven non synonymous mutations in L1 gene were recorded in patients from the Netherlands, while twenty one of these variations are located in the region that encodes for the immunodominant FG loop . Neverthdy titer . In addidy titer . Moreovedy titer . Finallydy titer ,48,86. NA previous analysis in the Indian population revealed a total of 20 non synonymous mutations, nine of which are located in the immunodominant loops including N56T (BC loop), T176N, V178G, A179T, N181T/I (EF loop), T266A, N285T (FG loop) and T353P (HI loop).Three-dimensional prediction models suggest that the amino acid mutations L158F, A179T, K236T, T266A, S296R, T353P, S396P, and K454T might influence the stability of the protein structure, while the amino acid mutations N56T, H76Y, N92T, T176N, V178G, N181T/I, N285T, T389S, K443Q, and L474F might prevent the structure of the L1 monomer .A more recent analysis that examined all the available complete and partial sequences of the HPV16 L1 gene demonstrated that the vast majority of nucleotide polymorphisms are accumulated in the DE (27.38%) and FG (31%) loops, while four mutations are frequently detected in other loops such as T176N (EF loop), N181T (EF loop), A266T (FG loop), and T353P/I/N (HI loop) . NotablyAlthough the L1 gene has been the focus of many studies, little is known concerning the variability of the HPV16 L2 gene. The role of the minor L2 capsid protein in viral pathogenicity is regarded as critical, since it is implicated not only in viral particle assembly and viral entry, but also ensures the efficient transport of viral DNA in the nucleus of infected cells and is involved in host immune response as well ,89,90. ACertain sequence variations within the HPV16 L2 gene are related to the different lineages. In particular, the A1–A3 sub-lineages harbor two variations ; the A4 sub-lineage harbors four variations ; the B lineage harbors 18 variations ; the C lineage contains 22 variations ; and the D lineage contains 28 variations  .The analyses of the HPV16 L2 gene in clinical samples are rather limited. However, it has been observed that the L2 gene is polymorphic, whereas the region that encodes for the N-terminal domain of the L2 protein was highly conserved. In particular, a previous analysis that was carried out in patients from Southwest China revealed nine non-synonymous nucleotide variations, while no sequence changes in the residues 65–78 and 108–120 were detected . In addiIn order to perform a sequence analysis of publicly available sequence data, we searched the NCBI taxonomy database for Human papillomavirus type 16  and retrieved 11,018 (10 June 2022) nucleotide records. These records were filtered based on the availability of country of isolation, thus resulting in 9658 sequences. These nucleotide sequences were subsequently scanned by the BLASTn algorithm  using thA global amino acid mutation analysis of HPV16 L1 revealed a total of seven high frequency amino acid substitutions  (63). The location of the respective mutations in the L1 protein as well as their distribution among different geographic locations is summarized in The large-scale sequence analysis of the L2 gene revealed that the specific L2 epitopes  appear to be conserved among the different HPV16 isolates globally . HoweverThe large-scale sequence analysis of the L2 gene revealed that the specific L2 epitopes  appear to be conserved among the different HPV16 isolates globally . HoweverThe residual mutations of the HPV16 L2 protein are detected in the central region and the C-terminal domain . NotablyΤhe US Food and Drug Administration (FDA) has approved a total of three prophylactic HPV vaccines based on the L1 protein. Until June 2020, 107 out of 194 WHO member states have started national HPV vaccination programs, whereas it has been estimated that in 2019 approximately 15% of girls and 4% of boys have been fully vaccinated . The WHOVarious amino acid substitutions have been recorded within or adjacent to the HPV16 L1 immunodominant loops ,85,86,88On the other hand, the L2 gene is considered as a polymorphic region, although its N-terminal domain is highly conserved ,101,103.It is of note that the amino acid changes that were found next to or within the immunodominant regions of L1 and L2 proteins are highly distributed in specific geographic areas. This variability might influence the potential of certain HPV16 strains to avoid immunological response in some populations, providing HPV16 strains with a selective advantage to establish persistent infection, thus leading to more severe dysplasia. However, further analyses of these amino acid changes need to be performed in order to confirm this hypothesis.Highly conserved regions of the HPV16 L1, L2 proteins have been extensively characterized, including the DC loop, DE loop of the L1 protein, the L2 specific epitopes and the N-terminal domain of the L2 protein ,101,103.To the best of our knowledge, this is the first extensive literature review and large-scale sequence analysis concerning the global variability of both HPV16 L1 and L2 genes. We presented important data concerning the variability of the late region of HPV16 DNA. Our observations suggest that the high prevalence of certain amino acid substitutions within or adjacent to specific immunodominant regions might influence the stability of the viral capsid as well as modify the ability of HPV16 in evading host—immune response in specific populations. The extensive mapping of amino acid substitutions in the HPV16 L1 and L2 epitopes should be cautiously considered in a given population.


Signed networks provide information to study the structure and composition of relationships (positive and negative) among individuals in a complex system. Individuals, through different criteria, form groups or organizations called communities. Community structures are one of the important properties of social networks. In this work, we aim to analyze the perturbation of negative relationships in communities. We developed a methodology to obtain and analyze the optimal community partitions in nine school networks in the state of Yucatán, México. We implemented a technique based on the social balance theory in signed networks to complete negative missing links and further applied two methods of community detection: Newman’s and Louvain’s algorithms. We obtain values close to Dunbar’s ratio for both types of relationships, positive and negative. The concepts of balance and frustration were analyzed, and modularity was used to measure the perturbation of negative relationships in communities. We observe differences among communities of different academic degrees. Elementary school communities are unstable, i.e. significantly perturbed by negative relationships, in secondary school communities are semi-stable, and in high school and the university the communities are stable. The analyzes indicate that a greater number of negative links in the networks does not necessarily imply higher instability in the communities, but other social factors are also involved. The field of social network analysis provides a formal way to define important social concepts and study relationships (links or edges) among actors (nodes). In the last years, network analysis has focused on unsigned networks (or networks with only positive links). Signed networks are defined as an extension of networks that include the additional information of positive and negative links. Many social networks allow positive and negative links , 2. In aCommunities in networks are defined as the groups of nodes within which the edges are dense but between which the edges are sparse . SociallSocial balance theory and frustration are two concepts that are linked with studies of signed networks . Social Properties at the community level may differ greatly from properties at a global network, which makes these studies very relevant to the field. In this work, we use nine real-world student social networks to find optimal community partitions and quantify the perturbation of enmity relationships among students. Consecutively, we raised the question: is the community partition of our networks a reflection of the real partition? We believe that communities that are not significantly perturbed by negative relationships match better communities in the real-system.The theory of signed communities is under development, most of the proposed approaches have been developed for communities having only positive edges. Our research unites the classical analysis of communities in unsigned networks with the theory of signed communities. We make use of three functions: Modularity, balance, and frustration. In addition, community partitions are analyzed using two methods of community detection: Newman’s (deterministic) , 20 and Lastly, social interactions in schools have been reported recently and had emerged has an important subject. In reference  the authThis study was approved by the Bioethics Committee for Research in Human Beings at Centro de Investigación y de Estudios Avanzados del IPN and we obtained written informed consent from the principal of the school that participated in this study. In addition, in elementary schools, the parents/guardians of each child who participated in the survey provided written informed consent. In secondary and high schools, the principals informed parents/guardians who provided verbal consent. On the day of the survey, it was explained to the students that they could decline or give their approval to save their answers. If the student agreed, the student signed an informed consent electronically at the end of the survey.The data set was collected through the application of surveys (May to October 2019). It is important to clarify that the surveys, although similar, were not applied in the same form. In three elementary schools , due to the unfeasibility of the use of electronic devices, the survey was applied in written form. In the rest of the schools, the survey was electronic, it was conducted using the Internet through a platform that we designed for that purpose. Data collection was supervised personally by professionals in the area, after a brief description of the necessary information for the research. All data was confidentially collected and anonymously analyzed. These data sets have been used in previous research by our work team –28.We designed and applied a questionnaire (survey) to students from nine schools. There are several studies in which the friendship relationships are represented by a diagram of concentric circles, where the smaller circles correspond to those friends who are closer to the person being analyzed, that is, in the first circle would be the best friends, in the next one, just friends and so on , 30. It A) were constructed. Original networks are directed signed networks. We consider that a positive reciprocal relationship between any two nodes (i.e. Aij = Aji = 1), is an indication of a strong friendship between students i and j, which we label a ‘best friend’ relationship. A one-way positive relationship we label a ‘friend’ relationship only. In an analogous way we treat the negative relationship as ‘worst enemy’ if Aij = Aji = −1. Just ‘enemy’ if only a one-way negative link exists.By extracting information from the surveys, the nine corresponding adjacency matrices  and an unbalanced one . Since there are more balanced triads, the reciprocal enmity link between nodes i and j is added.In the following, we present the procedure (heuristic) for adding the negative missing links in the networks . For this purpose, the social balance theory was used: negative links were added based on the idea that a common enemy of my friends is probably my enemy. To reduce the probability of adding negative links that do not exist in the real-system, a link was added only if one of the two students said he was an enemy of the other, that is if m+) and negative (m−) links in the networks after applying this methodology. 〈k〉+ and 〈k〉− represent the average degrees of best friend and worst enemy, respectively. Additionally, the m− column shows the percentages of reciprocal negative links in the networks. The difference among the node’s column and student’s column in After including the missing links  algorithms are two methods of community detection. In both methods, the number and size of the communities are unspecified but determined by the network organization. The aim is to create an optimal community partition on the network; that is, divide it only when a good subdivision exists and leave it undivided otherwise.Q+). It serves to compare the fraction of positive edges found inside communities in the observed network with a similar topological random network. Modularity is defined in δ = 1 if i and j nodes belong to the same community, and δ = 0 otherwise. The w+ symbols represent positive links. Networks with high modularity have numerous connections among the nodes inside a community and few connections among nodes of different communities  and diagonalizes it, obtaining its eigenvalues and eigenvectors, these indicate the ‘forces’ with which the nodes belong to one or another community. The aim is to find a partition that maximizes  in ref. .Q+. First, it finds small communities by optimizing locally the modularity for all nodes, then each small community is associated with a node and the process is repeated until convergence is reached, details can be found in ref. [On the other hand, the Louvain method computes the partition of the network and tries to maximize  in ref. . Louvain in ref. ). Good cQ− is defined analogously to Qs (Q-signed), is a linear combination of Q+ and Q− , see We use this concept to determine whether the communities we obtain are socially balanced. Here, the community social balance (CSB) is defined as the ratio of balanced triads in the communities  = 1 if i = j and zero otherwise [The frustration concept is based in two ideas. First, the signed community partition should have dense positive intra-connections and as many negative inter-connections as possible. Second, it should have as few as possible negative intra-connections and sparse positive inter-connections. Frustration is the opposite of these ideas, therefore, it is defined as the sum of positive inter-connections plus negative intra-connections, see therwise . We normThe student social networks that we are studying have two types of links  and both kinds of links should be analyzed as a whole thing. In reference  the authIn the present work, we ask ourselves if considering only positive links in the communities analysis, can lead also to some erroneous conclusions. We try to answer this question by implementing the following procedure and quantifying the results. For each of the nine schools, the signed network was built from the friendship and enmity relationships matrix. The positive links subnet was extracted and two community detection algorithms (Newman and Louvain) were applied to obtain the optimal communities partition. Added to this partition are the negative relationships that exist among its nodes; this is how we build signed communities. With this procedure we obtained an optimal partition created from positive relationships containing negative relationships that perturb their stability. The definition of modularity is used to measure this perturbation.Furthermore, we introduce the concepts of an unstable, semi-stable, and stable community and define them in the social sense. A community that has few antagonistic relationships is expected to have social stability, this means that it will have fewer conflicts among the individuals that integrate it. Whereas a community with many antagonistic relationships will be socially unstable.m−) in elementary and secondary schools is similar, while in high school and university this proportion is lower. It can also be noted that elementary schools have higher values 〈k〉+ and 〈k〉−, indicating that in these schools the students have more friendship and enmity relationships than in the rest of the schools. Socially, this could be because at an early age (6y to 12y) students do not have well-defined criteria for selecting their friends, while in adolescence and youth ages (from 12y), they are mature enough to form a better defined social circle. The same phenomenon occurs with enmities.k〉+ and 〈k〉− in elementary schools results in the formation of visible groups in signed networks. In The high values of 〈m− values . Similarly, λ = 6.31, λ = 10.00, and λ = 9.55 for the greedy method.The community distributions for each of the schools studied is plotted in We show four communities (Newman) from esRRC school  and fourIt would be expected that a small modification to the algorithms solves the problem of perturbed communities. However, this is not so simple, just removing nodes from a community or splitting a community into two causes the modularity function to decrease. Recent studies have addressed this problem through genetic and evolutive algorithms .In good partitions of signed communities, positive edges should be dense within communities and sparse between communities, and negative edges should be sparse within communities and dense between communities. From The signed communities obtained with the greedy method have a higher proportion (%) of negative links . HoweverThe difference in the value of frustration between Newman and greedy methods is almost constant (with exception of hsCPP). This is because students in signed networks initially have a fixed number of positive and negative relationships; thus, regardless of the optimal communities partition, the relationships will be the same. This phenomenon may indicate that there is a constant factor that related to frustration with the number of communities in which the school is divided, or more specifically, with their average size.The CSB is shown in It can be noted that the CSB values obtained by both methods are similar (except for hsHUN), this may indicate that the proportion of balanced triads in communities is independent (or nearly) of its optimal partition. This phenomenon is interesting because the CSB values would be expected to be different for different partitions. We believe this is due to signed networks having an intrinsic social balance factor that affects the CSB value. In other words, the number of balanced triads in the network significantly affects the CSB values of any optimal community partition. In conclusion, the social balance factor in systems like the ones we study in this paper provides important information on how balanced their communities are.Q+ values  and 0.9 (greedy). The latter indicates that the correspondent communities formed by positive relationships meet the community definition very well; i.e. their nodes are densely connected within communities and sparsely connected between communities. In this figure it can be observed that the greedy method optimizes these communities better.+ values . TheoretQs values , secondary schools have semi-stable communities, and in high schools and the university these are stable. We infer that the criteria for choosing relationships and forming social communities of students are better defined over time.The above indicates that primary school communities are formed with weak criteria (or even random ones). In secondary schools, communities begin to define themselves better and regain a certain degree of stability. Particularly in elementary schools, communities within the real-system could be very different from those generated by the algorithms when we only consider positive links. On the other hand, in the high schools and university, the communities are a very good approximation of the communities in the social system that we study.Here, we consider the smallest size for a community is two nodes. This consideration initially generated uncertainty because in some networks there are too many two-node communities see  and it iTo better understand friendship and enmity relationships, in the applied surveys, we asked questions about the meaning of these concepts. The responses showed that both concepts have extensive meaning, and include different elements of perception. However, young adults report the greatest consistency in the perception of relationships. Whetnall  and BurkAt the beginning of the research, we expected that the PPC value was directly related to the percentage of negative links in the networks, yet this is not the case. Secondary school networks have high percentages of negative links , but theThe PPC value  shows a The relationship between balance and stability has been studied in the field of signed communities. Sun et al. defined that a community is stable if all the triads in it are balanced . Here, wO(n * log(n))) [We chose two different methods to detect communities. Although the Newman method results in more communities , 47, andlog(n))) . The netQ+) in elementary (and some secondary) school’s communities are the consequence of random relations. The question is, if we have only positive links, how do we know if the modeled communities are a reliable representation? We infer that if individuals have well-defined concepts, we will obtain stable communities and these will be a reliable representation of the real-system.Studies on signed communities define that most of the negative links should be observed between communities, with very few negative links within communities. In other words, social communities must be stable. We simulated communities where only positive links were present and obtained communities where the definition of signed communities is not met. We believe that these communities do not represent the real-system. It is not our intention to suggest that the community detection algorithms are erroneous; these methods have been validated in various articles with both, real (one of the best known is the Zachary’s karate club ) and synNegative hubs were found in the communities see . AlthougFinally, there are methods (e.g. machine learning ) that alWe conducted surveys on friendship and enmity relationships among students from nine schools of different academic grades and built signed networks. A methodology to obtain signed communities and quantify the negative links perturbation in the optimal communities partitions was developed. We obtained Dunbar’s ratios for both types of relationships, positive and negative, and for all the nine schools studied. When analyzing the PPC values, it was observed that a greater number of negative links in the networks does not necessarily imply greater instability in the communities. We also conclude that the academic degree (age) is related to the viewpoint of the concepts of friendship and enmity; and that this influences the stability of the communities.S1 FileQuestions asked in the data collection (english and spanish versions).(PDF)Click here for additional data file.S1 TableTables with the numerical values of friendship and enmity relationships in school networks.(PDF)Click here for additional data file.S1 DatasetSigned adjacency matrices used in the study (.cvs).(ZIP)Click here for additional data file.S1 FigFigures showing the variability of the Louvain method.(PDF)Click here for additional data file.