The aim of this study was to evaluate the relationship between personality traits and perceived treatment success in oral lichen planus.A total of 53 patients with diagnosed oral lichen planus were evaluated at the time of diagnosis and along the course of their treatment. The visual analogue scale (VAS) was used for evaluating pain and burning sensation, along with an evaluation of the oral health-related quality of life (OHIP) and the clinical severity. In order to determine the personality trait, the NEO-FFI questionnaire was applied. Data were assessed with the statistical software Stata by a multiple linear regression.P < 0.05).A significant relationship between the two personality traits: “conscientiousness” and “extraversion” and a perceived improvement in oral lichen planus could be observed. The higher the “conscientiousness,” the better the perceived oral health-related quality of life. Furthermore, “extraversion” had a significant influence on the improvement in clinical severity index (Personality traits, especially conscientiousness and extraversion, have a significant impact on the perception of therapeutic intervention in oral lichen planus.As personalized patient management is gaining importance and psychosocial factors play a significant role in mucosal diseases, the patient’s psychological profile should be considered in the oral lichen planus management. Oral lichen planus (OLP) is a chronic inflammatory mucocutaneous disease. Prevalent in around 2% of the general population, it mainly affects women between 30 and 60 years of age. This immune disease is clinically characterized by different types of lesions, like reticular, so-called Wickham Striae, atrophic, or erosive areas, which may alternate . PatientOLP is assumed to be an autoimmune disease, yet the exact etiology is unknown. Psychological factors could play an important role as emotional stress is likely to be an associated risk factor. Furthermore, the burdens of chronic disease and persisting pain patients suffering from OLP are faced with require copying strategies. Personality traits influence behavior, compliance, and the perceived health-related quality of life, in mucosal diseases as elsewhere , 9. WhetThe consequences of this condition on daily life, coping with the disease and quality of life, are all reflected by the Oral Health Impact Profile (OHIP). Also, the responsiveness of OHIP scores under therapy has been evaluated –16.Clinicians should consider that psychological traits might play a relevant role in establishing individual limits of quality of life for specific treatments. Some measurable personality traits seem to have a considerable impact on individual satisfaction with therapy .Since great importance is currently given to subjective outcome parameters, this study ought to provide additional data augmenting the possible contribution to clinical intervention.We hypothesize that some personality traits are associated with higher scores of oral health-related quality of life in patients with OLP undergoing treatment.This was an observational cohort study carried out at the University Clinic of Dentistry of Vienna, Austria. Ethical approval was obtained from the Ethics Committee of the Medical University of Vienna. All patients gave written consent for their participation. Patients were eligible, if their diagnosis of oral lichen planus (OLP) was confirmed by histopathology and direct immunofluorescence assay; any removable dentures were correctly fitting; minimum age was 18 years; no nicotine abuse was reported. Patients were excluded with a malignant transformation, severe dysplasia in histopathology, carcinoma in situ, severe vitamin deficiency, pregnancy, age below 18 years, lactation period, nicotine abuse, the presence of asymptomatic OLP, or oral mucositis of other origins .Patients with OLP were recruited at the outpatient clinic for mucosal diseases of the Division of Oral Surgery between October 2015 and August 2016. The diagnosis of OLP was made according to the clinical appearance and further confirmed by histopathology and direct immunofluorescence (IFT) analysis. All the tissue samples were taken by one specially trained surgeon (GD) and a specialist (KR) in immune dermatology and mucosal histopathology performed these analyses. The type of treatment , topical triamcinolon (Volon® A), tacrolimus, cyclosporin, photodynamic therapy (PDT), systemic glucocorticoids or deproteinized hemodialysate of dairy calves (Solcoseryl® ointment), the time between appointments, the clinical severity of OLP, and the subjective outcome parameters of oral health-related quality of life and pain intensity were assessed as well as the personality traits by questionnaires. The type of treatment was chosen individually depending on the patient’s symptoms in accordance with the treating dermatologist. In cases where the first-line treatment was not effective and patients came for a second opinion, calcineurin inhibitors were prescribed by a dermatologist (KR). Some of the patients suffering from lichen ruber planus of the skin or in other mucosal locations received systemic glucocorticoids. In mild cases or when patients refused immunosuppressant therapy, hyaluronic acid or hemodialysate was prescribed. All treatment options were discussed with the patient and the treating dermatologist, if available. Patients were recalled according to the clinical presentation every 3–6 months (90–200 days). In cases of subjective worsening, patients had the possibility to seek for an earlier appointment.The clinical presentation and the therapeutic improvement of clinical signs may be assessed by a clinical criteria score. The Thongprasom score is such a clinical severity index evaluating disease intensity in many OLP studies . The CSI2; 3, white striae with atrophic area > 1 cm2; 4, white striae with erosion < 1 cm2; 5, white striae with erosion > 1 cm2.0, no lesion/normal mucosa; 1, mild striae/no erythema; 2, white striae with atrophic area < 1 cmSubjective pain intensity evaluation is widely performed by a visual analogue scale (VAS) . A custoThe OHIP is a cross-culturally adapted oral health-specific outcome measure recognized as an instrument suitable for the assessment of the oral health-related quality of life in cross-sectional as well as longitudinal studies. The Oral Health Impact Profile questionnaire German version (OHIP-G) is an oral health-related quality of life instrument containing 53 questions concerning the previous 4 weeks of the patients’ life. Answers to the 53 items of the OHIP-G are given on a Likert-type five-point scale . In this analysis, the total summary OHIP-G score was utilized, calculated by summing the score of the answers to each question. Higher scores indicated the lower oral health-related quality of life. Summing the responses to subsets of items created subscale scores. OHIP-49 is divided into seven different parts. The possible score range for each of these is as follows: “functional limitation” (9 items)—from 0 to 36; “physical pain” (9 items)—from 0 to 36; “psychological discomfort” (5 items)—from 0 to 20; “physical disability” (9 items)—from 0 to 36; “psychological disability” (6 items)—from 0 to 24; “social disability” (5 items)—from 0 to 20; “handicap” (6 items)—from 0 to 24; and finally “overall OHIP score” (49 items)—from 0 to 196. The German version has additional questions “DT” (4 items)—from 0 to 16. Four dimensions  served as principal components of oral health-related quality of life. The dimensions were calculated according to 21 stable rotational components as described previously.The OHIP-G was assessed at each appointment.This is an instrument to determine personality traits containing 60 questions. It analyzes five main behavior domains . Each of these domains is examined through 12 questions to be answered by one of the following Likert-type answers: “strong disagreement,” 0; “disagreement,” 1; “neutral,” 2; “agreement,” 3; or “strong agreement,” 4.Data processing and scoring procedures were performed using appropriate software provided with the instrument forms. Scores were calculated by specific algorithms based on the population characteristics . The average ratings for each domain in NEO-FFI-R were related to OHIP scores to verify the influence of psychological profiles.f2 = 0.15, statistical power of 0.85, 17 predictor variables , and a probability level P = 0.05 at least 51 patients needed to be included for the multiple regression analyses. A multiple repeated measure regression (repeated over one to four treatment points per patient) was used to establish a predictive model on oral health-related qualitative life with oral lichen planus.For a medium effect size of P < 0.05 and highly significant when P < 0.001.The repeated measure regression was performed with Stata statistical software package version 15 (Stata Corp LCC). Statistical values were considered significant when The study comprised a total of 53 patients, 46 women (86.8%), and 7 men 13.2%), distributed in following age categories: 21–35 years of age 6 patients, 36–50 years of age 12 patients, 51–65 years of age 19 patients, 66–80 years of age 16 patients. The mean age was 56.5 ± 13.7 years (min. 21–max. 79). The characteristics of the collective are presented in Table 3.2%, disn = 42), 18.9% (n = 10) were wearing a partial denture, and 1.9% had a complete denture (n = 1). The mean number of missing teeth was 5.3 ± 5.5 teeth, 18.9% (n = 10) had no missing teeth, and 3.8% had 20 missing teeth (n = 2). The mean time between the first visit and the last visit was 384 ± 351 days (min. 14–max. 1713 days). The mean time between the first and second visit was 241 ± 220 days (min. 14–max. 945 days). Eighteen patients came for a third visit after a mean period of 269 ± 219 days between the third and second visit (min. 14–max 959). Whereas 7 patients came for a fourth visit after a mean period of 392 ± 413 days between third and fourth visit (min. 91–max. 1234). Eleven patients came before the recommended recall period and 32 after (90–200 days).No removable denture was used by 79.2% (The difference between the last and first appointment was as follows:P = 0.02).The mean VAS improved by − 15 ± 29 mm, men showing significantly more improvement  improved by − 1.2 ± 1.2 under therapy. CSI improved significantly by the following factors: age, the number of missing teeth and the personality trait “extraversion.”In summary for all four appointments, CSI 0 was counted 6 times: CSI 1, 37 counts; CSI 2, 33 counts; CSI 3, 21 counts; CSI 4, 19 counts; CSI 5, 1 count. At the last visit, CSI 4 and 5 were not present while found in 15 patients at the baseline visit (Table P = 0.04)  Tables , when hy) Tables .Table 4Dn = 46) of the patients were using hyaluronic acid (Gengigel®) for a mean of 313 ± 350 days three times a day, 52.8% (n = 28) topical triamcinolone for a mean of 162 ± 321 days three times a day, 7.5% (n = 7) topical tacrolimus for a mean of 21 ± 88 days twice a day, 5.7% (n = 3) systemic glucocorticoids for a mean of 18 ± 127 days, 22.6% (n = 12) Solcoseryl dental adhaesive® several times a day for a mean of 69 ± 198 days, 3.8% (n = 2) cyclosporine mouth rinse (200 mg/twice daily for five minutes) for 19 ± 115 days, and 3.8% photodynamic therapy with 5-aminolevulinic acid once a day for 2 ± 11 days.Considering the different treatment options, 86.8%  influenced the OHIP-G summary score, especially the dimensions “physical pain,” “physical disability,” “functional limitation,” “psychological disability,” “social disability,” “handicap,” “psychological discomfort,” “orofacial pain,” “oral function,” “psychosocial impact,” and “appearance.” The worse the clinical presentation is, the higher the impact on perceived daily quality of life Table .Hyaluronic acid (Gengigel®) had a significant influence on OHIP improvement, subscore “physical pain,” “psychological discomfort,” “physical disability,” “psychological disability, dimension,” “psychosocial impact, dimension,” “orofacial pain,” and “appearance.”Topical corticosteroids had a significant negative impact on OHIP-G, “functional limitation,” “psychological disability,” “physical disability,” “psychological discomfort,” “physical pain,” “DT,” and “psychosocial impact.”Systemic corticosteroids had a negative impact on OHIP-G, subscores “handicap,” “physical disability,” “oral function,” and “functional limitation.”P = 0.04).The photodynamic therapy had a significant influence on the perceived worsening of “oral function”  during the course of treatment.A pronounced “extraversion” personality trait significantly influenced the clinical presentation assessed by CSI  questionnaire evaluates five personality traits  and consists of a psychological test with good structure and reliability. The trait “conscientiousness” had a significant influence on oral health-related quality of life in physical and psychosocial aspects. This trait is characterized by a tendency to be organized, show self-discipline, act dutifully, aim for achievement, and prefer planned rather than spontaneous behavior. Also, in recent literature, conscientiousness was positively related to treatment adherence, as this trait estimates motivation in goal-directed behavior. Regularity is required in topical treatment with immunomodulating agents. Therefore, the facet “conscientiousness” could influence higher treatment adherence. Furthermore, the caregiver’s conscientiousness could have a significant influence on treatment outcome . Self-efThe trait “extraversion” had a significant influence on the clinical severity of OLP patients. Extraversion is characterized by gratification from the external environment of the patient, linking interaction with others, being full of energy in the presence of others, and prefers human companionship to being on their own. Whether chronic disease may influence personality traits is a matter of debate. Nevertheless, clinically more severe cases may reduce the tendency to seek stimulation in the company of others and talkativeness. “Extraversion” seems to be protective in the psychosocial domain. This trait includes characteristics such as excitability, sociability, assertiveness, and high amounts of emotional expressiveness. Thus, an improvement in clinical severity may influence the sociability and talkativeness of patients. Vice versa, “extraversion” may modulate cortisol response to stress in chronic disease. It is known that OLP can appear and worsen during stressful events. Psychosocial factors such as personality traits are significantly associated with quality of life ratings. Such associations should be considered when the quality of life measurements are used and interpreted. Certain personality traits can lead to predicting positive health outcomes. In an elderly Japanese sample, conscientiousness, extraversion, and openness were related to a lower risk of mortality . IndividA recent Cochrane review  found noWe must also consider that many patients have prejudices again corticosteroids and patients as well as doctors have not been blinded to the prescribed treatments in the present study. This could have affected the regularity of the application. Furthermore, a strong weakness of the present study is the lack of a control group receiving a placebo or no treatment.Topical steroids remain the first-line treatment for symptomatic OLP with proven efficacy and safety, but topical agents can probably not remain on the oral mucosa sufficient time to be absorbed. Therefore, the systemic or intralesional application is indicated when topical corticosteroid treatment is insufficient. Corticosteroid treatments nevertheless raise concerns in many patients due to the possible side effects . One sidA recent study showed that 0.2% hyaluronic acid effectively improved the symptoms associated with OLP even after cessation of treatment. Hyaluronic acid is an effective substitute for topical corticosteroid with similar results. Moreover, in severely painful OLP, 0.2% HA could be used in addition to other topical drugs, such as corticosteroids and calcineurin inhibitors, to reduce the overall amount of immunosuppressant therapy used in a course of treatment .Additionally, topical treatments depend on the patient’s compliance, which may influence results. As mentioned previously “conscientiousness” correlates with self-discipline and could influence compliance. The personality factors “conscientiousness” and “agreeableness” were presented as predictors of high treatment adherence in oncology, recently . ResultsConcluding, “conscientiousness” and “extraversion” have a significant influence on therapeutic outcomes in OLP patients mainly treated with different topical agents.Assessment of personality factors may be helpful in predicting the patient’s cooperation and may affect treatment. Therefore, it would be reasonable to assess personality traits, in order to be more aware of patients’ expectations and their possible compliance with an offered treatment. This may avoid extra time and effort. Psychological facets play a role in OLP management and explain the impacts on daily living and patients’ satisfaction.


Overall, the two genotypes appear to share similar defense mechanisms but, in the short term, “Bianco Avorio” depends mainly on non-enzymatic antioxidant phenylpropanoids for ROS scavenging, while “Spagnolo” maintains a larger arsenal of defenses. Upon prolonged stress, proline could have contributed to protection of metabolic functions in both genotypes. Our results provide cues that can be exploited for cardoon genetic improvement and highlight genotypic differences for breeding salinity tolerant varieties.Cultivated cardoon is a multipurpose crop with adaptability to limiting environments. Two genotypes (“Bianco Avorio” and “Spagnolo”) were comparatively characterized in response to short and prolonged 100 mM NaCl stress in hydroponics. Salt induced no growth variations between genotypes or symptoms of NaCl toxicity, but boosted ABA accumulation in roots and leaves. Both genotypes had high constitutive phenol content, whose major components were depleted upon 2 days of stress only in “Bianco Avorio”. Prolonged stress stimulated accumulation of proline, phenylpropanoids, and related transcripts, and non-enzymatic antioxidant activity. Decreased antioxidant enzymes activities upon short stress did not occur for APX in “Spagnolo”, indicating a stronger impairment of enzymatic defenses in “Bianco Avorio”. Nonetheless, H Excessive soil salinization is particularly increasing in arid and semi-arid cultivation areas, where evapotranspiration is more prominent than rainfall. In this scenario, salinity is becoming one of the most serious abiotic stresses affecting plant growth and development and hence crop productivity . High saavenging ,4,5. Altavenging ,7 major avenging ,8. Thereavenging . Neverthavenging ,11. Polyavenging ,13. Phenavenging . MoreoveHence, tolerance derives from a tight and efficient interaction between biochemical signals, which act in both additive and synergistic ways in the plant response to salinity. In this regard, investigation of mechanisms of salt resistance/tolerance has been pursued in several species for the development of salt-tolerant genotypes, thus opening the possibility to use salinized land for crop production.Cynara cardunculus L. var altilis, the cultivated cardoon, is a perennial species native to the Mediterranean region. Cardoon requires low nutrients and nitrogen and is usually cultivated in arid and semi-arid environments, where soil salinity can be remarkable. Several studies highlighted the highly adaptive potential of cardoon, focusing on its ability to face abiotic stressors such as salt, drought and heavy metals [y metals ,19,20,21y metals ,23. Alony metals . Cynaropy metals ,26. Besiy metals ,28. In ay metals ,30 in an+ as less toxic than K+ and, conversely to most of the glycophytes, counteracted osmotic unbalance through regulation of inorganic ion content, translocating Na+ excess to the shoots. Most probably, cardoon is able to tolerate Na+ through a tissue tolerance mechanism, although this species might use also other mechanisms to overcome saline stress. In accordance, more recently [References ,31 reporrecently  showed tStudies on the potential of Italian commercial cardoon genotypes focused on production of biomass, energy or other valuable products, also with very low agronomical inputs ,33,34. BSince “Bianco Avorio” and “Spagnolo” appear very promising and particularly suited for cultivation under low input management in temperate climates, we characterized these cardoon genotypes for both their performances under severe saline stress conditions and tolerance mechanisms. In the present work, the physiological, transcriptional, enzymatic and metabolic changes determined by short and prolonged NaCl stress imposition on the two cardoon genotypes “Bianco Avorio” and “Spagnolo” were analyzed in order to assess the traits mostly involved in saline tolerance. Our study highlights the metabolic investment of cardoon plants to face salt stress and provides new evidence on how cardoon plants use their antioxidant machinery to prevent oxidative damage caused by growth in saline environments. Overall, this study delivers new findings about cardoon saline tolerance and may supply useful cues in designing strategies for breeding improved genotypes for sustainable cultivation.−1) in hydroponic conditions for up to 21 days (−1) plants of both genotypes. Shoot biomass showed no significant differences between the two genotypes and in comparison with their respective controls in terms of dry weight, though a significant decrease in shoot fresh weight was detected in both genotypes. Stimulation of root growth was detected in both genotypes, with an increase of specific root length of about 4% in “Bianco Avorio” and 12% in “Spagnolo” under NaCl stress  of leaves , evaluatp-coumaroylquinic acid, pCoQA; feruloylquinic acid, FQA), dicaffeoylquinic acids , and flavones  [−1 DW of caffeoylquinic derivatives (CQAs) and flavones, respectively, whereas in “Spagnolo” they were about 4 times less abundant. Upon 2 days of saline stress a depletion (about 50%) of phenylpropanoids and flavones was observed in “Bianco Avorio”, whereas in “Spagnolo” leaves the level of CQAs remained comparable to controls. After 21 days, a strong reduction of both phenolic classes was detected in unstressed leaves of the two investigated genotypes . However, the long-term saline stress induced a significant increase in their amount in both genotypes . In detail, at 21 days of saline treatment, the caffeoylquinic acids pool increased more than thirty times above the control in both genotypes, though slightly more in “Bianco Avorio”. Similarly, a significant increase in the levels of luteolin and its glycosides was detected in both genotypes.Reference  reportedenin, A) ,36,37 anenin, A)   MYB12, known as regulator of phenylpropanoid biosynthesis, in cardoon unstressed and 21 days NaCl-stressed leaves. Alongside, we investigated key genes involved in sesquiterpene biosynthesis, namely germacrene A synthase GAS, germacrene A oxidase GAO and custonolide synthase COS.The relationship of transcriptional abundance of key phenylpropanoid and sesquiterpenes biosynthetic genes with metabolic changes within each genotype and treatment was further investigated. Namely, variation in the transcriptional activities was monitored at 21 days of saline stress imposition, when the largest metabolic changes were observed. We examined expression levels of Hydroxycinnamoyl transferase HCT HQT and FNSII, F3′H, respectively, were upregulated by NaCl stress in both genotypes to a similar extent. A significant accumulation of MYB12 transcripts was detected both in “Bianco Avorio” and “Spagnolo” stressed leaves, although significantly higher in “Spagnolo” . Upon NaCl stress, no changes were detected in “Spagnolo”, while a significant reduction to about 0.3 mmol TEAC g−1 DW was observed in “Bianco Avorio”, whose antioxidant capacity was nonetheless significantly higher than “Spagnolo”. After 21 days, the antioxidant capacity of control leaves was much lower than after two days and was not different between genotypes, but it increased almost three times upon salt stress in both genotypes. As a non-enzymatic antioxidant, we measured proline content in leaves during short and prolonged stress (−1 FW) and six (≈4 µmol g−1 FW) times increase respect to controls in “Bianco Avorio” and “Spagnolo” leaves, respectively.The trend observed for TP was reflected by the changes in antioxidant capacity a. In thed stress b. At 2 dTo maintain metabolic efficiency and hence normal growth and development, a balanced ratio between production and detoxification of oxygen radicals is necessary, and more so under stress. Therefore, antioxidant enzymes are often reported to play an important role in imparting tolerance in plants under saline conditions. To investigate the ROS scavenging ability of “Bianco Avorio” and “Spagnolo”, we measured the activities of superoxide dismutase SOD, catalase CAT, and ascorbate peroxidase APX, during short and prolonged NaCl stress . Upon 2 2O2 production in “Bianco Avorio” and “Spagnolo” leaves  produced under stress conditions, we measured malonyldialdeide MDA and hydrogen peroxide H” leaves a,b. Both” leaves a. Namely+ excess to older leaves would have caused leaf yellowing and/or dropping and lack of photosynthates exportation to young leaves would have determined growth reduction of new leaves [Cultivation in saline soils or irrigation with low quality saline water, instead of the increasingly demanded fresh water, can contribute to a more sustainable agriculture. For these practices to become more widely diffuse, it is necessary to evaluate the effects of saline stress on plant growth and development as well as to elucidate the mechanisms of salinity tolerance/sensitivity, in order to design breeding strategies for salt resistant varieties. Cardoon is classified as a species well adapted to moderately high salt concentration . Neverthw leaves . The invw leaves . Indeed,w leaves , we detew leaves . An incrw leaves . In anotw leaves . Overallw leaves , even atPlant perception of a stress status, mainly water deficit and salt excess, results in ABA biosynthesis , which iSecondary metabolites, especially phenylpropanoids, accumulate in plants in order to counteract oxidative damage induced by abiotic stressors  Our metaExpression profiling of biosynthetic and regulatory genes involved in phenylpropanoids and sesquiterpenes formation at 21 days of salt stress, when most metabolic changes occurred, supported the metabolic trends. Salt stress significantly increased the expression levels of phenylpropanoid-related genes and of Myb12 TF in both genotypes. This MYB TF is a regulator of flavonol production  and its Although different plants may use individual strategies to fine-tune defensive responses under stress, quite often there is a close relationship between proline accumulation and stress tolerance. Accumulation of compatible solutes  is a common mechanism for plants to cope with stress by maintaining cellular turgor . In addi2O2, were not different in NaCl-treated leaves compared to their respective controls, confirming that both “Bianco Avorio” and “Spagnolo” were able to tolerate the stress levels applied in this study.It is reported in several species ,56 that Although the results need to be tested in field conditions, where other factors such as soil pH may have significant effects, they corroborate the idea that severe NaCl conditions did not impair growth and development of “Bianco Avorio” and “Spagnolo” and that phenolic antioxidants and proline more than antioxidant enzymes could serve as main tolerance mechanism in these cardoon genotypes. Albeit we report that both genotypes are tolerant to NaCl treatment, however “Spagnolo” is able to better tolerate stress by maintaining the integrity of crucial biochemical functions that could impart defensive advantages on the long term.Cynara cardunculus L. var. altilis cv. “Bianco Avorio” and “Spagnolo”, kindly provided by La Semiorto Sementi, Lavorate di Sarno, Italy) and Arca 2010 , respectively, were germinated in soil and transplanted after one month into hydroponics containers into a temperature-controlled greenhouse of the CNR-IBBR Portici (27 °C day/24 °C night). Each experimental unit consisted of 48 plants of each genotype per treatment in a completely randomized block design. Plants were kept in half-strength modified Hoagland nutrient solution  for about 7 days, followed by 7 additional days in full-strength modified nutrient solution (electric conductivity of 1.92 mS cm−1) as described by [−1) and maintained for 21 days. The pH of the nutrient solutions was 6.0 ± 0.3. All nutrient solutions were prepared using analytical grade chemicals and deionized water. To prevent large fluctuations in EC, pH, and ionic concentrations, the nutrient solutions were completely renewed from all tanks weekly. Within each treatment and genotype, 16 randomly selected plants were harvested for short saline stress measurements at 2 days of 100 mM NaCl imposition, and 16 plants were used for prolonged stress measurements after 21 day of 100 mM NaCl imposition. The remaining 16 plants per thesis (genotype × treatment) were used for destructive biometric parameters analysis, as below described.Seeds of ribed by ,57. EachFor RNA extraction and biochemical analyses, for each genotype, treatment and time point, four biological replicates consisting of four plants were collected. Leaves and roots were harvested in liquid nitrogen and frozen at −80 °C.For growth evaluation, 16 cardoon plants for each genotype and treatment were harvested at 21 days, recording the width of the youngest fully-expanded leaf (last leaf), and shoot and roots fresh weight. Shoot and root tissues were then dried in a forced-air oven at 60 °C for 72 h for dry biomass determination (DW). Root DW/shoot DW ratio (R:S) and specific root length, corresponding to the total root length/root DW (SRL) were measured according to Leaf greenness was measured using a portable chlorophyll meter soil plant analysis development (SPAD)  in plants exposed to 2 and 21 days of 0 mM (control) or 100 mM NaCl (stress treatment) treatment. SPAD readings were the average of 3 measurements from at the central point of the leaf between the midrib and the leaf margin of the third leaf starting from the top. For each genotype/treatment, SPAD values are expressed as mean ± SD of ten plants.g for 10 min, quantitative free ABA determination was performed on three biological triplicates and technical duplicates by the competitive ELISA Phytodetek ABA test kit  following the provider’s protocol. Color absorbance was read at 405 nm using a plate auto-reader  and free ABA content was expressed in nmol g−1 FW.For ABA extraction, freeze-dried root and leaf samples (150 mg) were homogenized in liquid nitrogen and incubated in 2 mL of water overnight at 4 °C. After sample centrifugation at 9300× v/v) and a matrix/solvent ratio of 1:20. The pooled extracts were dried under a gentle nitrogen flow. Finally, the residues were reconstituted with 1 mL of aqueous methanol (50% v/v) and filtered through 0.20 μM regenerated cellulose filters , before the chromatographic analysis. Dilutions of the extracts were used for determination of total phenol content and for ABTS assays, as reported below.Exhaustive extraction of the cardoon leaf phenolic fraction was performed according to . In deta2CO3  were added in each well and the reaction mixtures were incubated at 25 °C for 45 min. Absorbances were read at 725 nm with a Multiskan Go microplate spectrophotometer . Gallic acid (GA) was used as reference standard and TPC was estimated from the GA calibration curve . The results were expressed as GA equivalents per 100 g of dried leaf .TPC of cardoon leaves was determined using the Folin–Ciocalteu colorimetric method according to . BrieflyTo determine the qualitative profile of cardoon metabolites, the characterization of leaf extracts was performed by UHPLC-DAD-HRMS. Analyses were carried out using a PLATINblue UHPLC system  equipped with a Kinetex C18 column  and connected to a LTQ OrbiTrap XL mass spectrometer  operating in negative ionization mode with an ESI source. The chromatographic and mass spectrometer conditions were the same as those reported by . Metabol−1). For diCQAs , 1,3-diCQA was used as reference standard. Cynaropicrin levels in leaf samples were estimated related to UV response. The levels of luteolin and its glycoside derivatives was estimated using MS detection in MRM mode , due to their low and variable levels in the different samples that not allowed the UV quantification. Luteolin glycosides amounts were calculated using calibration curve of luteolin-7-O-glucoside. The amount of the compounds was finally expressed as mg per 100 g of dried leaf .5-CQA, 1,5- and 3,5-diCQA, luteolin glycosides , luteolin and cynaropicrin were detected in significant quantities in all the different leaf extracts. Their contents in extracts were determined by UHPLC-UV  and UHPLC-MS (luteolin glycosides) analyses. The chromatographic conditions were the same as those used for UHPLC-DAD-HRMS analysis. In the UHPLC-UV analysis, the chromatograms were recorded at 325, 350, and 220 nm for the detection of caffeoylquinic acids, flavones, and cynaropicrin, respectively. External standard calibration method was used to quantify the main phenolic components of cardoon leaves , according to previously validated method [® SYBR® Green qPCR Super Mix  in a ABI7900 HT . Each PCR reaction (20 μL) contained 10 μL real-time qRT-PCR Mix, 4 μL of a 1:25 dilution of cDNA and 0.25 μM of each specific primer designed on sequences retrieved either from X. The thermal cycling conditions were 50 °C for 2 min, 95 °C for 2 min, followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C. PCR product melting curves were analyzed for the presence of a single peak. All reactions were performed on biological triplicates and technical duplicates and fold change measurements calculated with the 2−ΔΔCT method [Leaves of cardoon plants exposed to 2 days and 21 days of 0 or 100 mM NaCl were collected for RNA extraction and biochemical analyses. Samples were ground in liquid nitrogen and total RNA was extracted from 100 mg of tissue using the RNeasy kit . After the visual check of RNA integrity on agarose gels and quantification using a NanoDrop ND-8000 spectrophotometer , 1 μg of total RNA was reversed transcribed using the QuantiTec Reverse Transcription Kit , according to the manufacturer’s instructions. Real-time qRT-PCR was performed with PlatinumT method . Each ge•+ solution (about 1 mM) was mixed with 5 μL of diluted extracts (five dilutions with approximately 20–80% control absorbance), Trolox  reference standard solutions or PBS (control), and 300 μL of the mixtures were transferred into a 96-well plate. The absorbance was measured at 734 nm, after 60 min of incubation in the dark at 30 °C, with a microplate spectrophotometer reader Multiskan Go . Trolox was used as reference standard . ABTS assay results were expressed as TEAC per 100 mg DW of extract .The ABTS scavenging capacity assay was carried out in 96-well plates according to . Brieflyv/v), then 50 µL of extracts were mixed with 100 µL of acidic ninhydrin reagent (ninhydrin 1% (w/v) in acetic acid 60% (v/v), ethanol 20% (v/v)), following a procedure previously described by [−1 FW) using three biological and three technical replicates.For Proline determination, fresh leaf samples (250 mg) were homogenized in liquid nitrogen and extracted with 70:30 ethanol:water mixture  standard curve (in the range 0.25–2 mg ml−1) and expressed in mg g−1 FW according to Bradford assay [−1 protein. Catalase  activity was measured at 240 nm, determining the rate of conversion of H2O2 to O2 and water, as described by [2O2 min−1 mg−1 protein. Ascorbate peroxidase  activity was determined following the H2O2-dependent oxidation of ascorbic acid (ASA) at 265 nm in a reaction mixture composed of 50 mM ASA, 90 mM H2O2, 20 µL protein extract and 0.1 M phosphate buffer (pH 6.4) [2O2-dependent ASA oxidation and the H2O2-independent ASA oxidation. APX activity was expressed as µmol ASA min−1 mg−1 protein. All the assays were performed in three biological and two technical replicates in 96 well plates and were adapted to small volumes. The amount of proteins was calculated.Antioxidant enzyme activities were measured in leaves sampled at 2 days and 21 days, as previously reported. Fresh leaf material (100 mg) was rapidly frozen in liquid nitrogen and extracted with 0.5 mL of 100 mM potassium phosphate buffer (pH 7.0) containing ethylenediamine tetraacetic acid (EDTA). The extracts were then centrifuged at 11,000× rd assay . Spectroribed by . CAT enz(pH 6.4) . The actg for 10 min at 4 °C. A 0.5 mL aliquot of the supernatant was mixed with 1.5 mL of 0.5% TBA prepared in TCA 20% and incubated at 90 °C for 20 min. After stopping the reaction in an ice bath, samples were centrifuged at 10,000× g for 5 min. The supernatant absorbance at 532 nm was then measured. After subtracting the non-specific absorbance at 600 nm, MDA concentration (three replicates per treatment) was determined using the extinction coefficient 155 mM−1 cm−1. H2O2 was determined by measuring potassium iodide (KI) oxidation by H2O2 in acidic medium according to [w/v) TCA. The homogenate was centrifuged at 12,000× g for 15 min and 0.25 mL of the supernatant was added to 0.25 mL 10 mM potassium phosphate buffer (pH 7.0) and 0.5 mL 1 M KI. The absorbency of supernatant was read at 390 nm. A calibration curve obtained with H2O2 standard solutions prepared in 0.1% TCA was used for quantificationLipid peroxidation was estimated by determining the malonyldialdehyde (MDA) contents in leaves. One hundred milligrams of dried samples were homogenized in 2 mL of 0.1% TCA. The homogenate was centrifuged at 15,000× rding to . Cardoonp < 0.05 and the Tukey’s post hoc test was applied for mean separation. SigmaStat version 11.0 software was used to perform statistical comparisons.Results are presented as mean ± standard deviation (SD). The statistical significance of data was evaluated through one-way analysis of variance (ANOVA). Statistical significance was set at


Bacillus cereus group bacteria including biofilm formation and motility, and CdgF was previously identified as a diguanylate cyclase promoting biofilm formation in B. thuringiensis. C-di-GMP can exert its function as a second messenger via riboswitch binding, and a functional c-di-GMP-responsive riboswitch has been found upstream of cbpA in various B. cereus group strains. Protein signature recognition predicted CbpA to be a cell wall-anchored surface protein with a fibrinogen or collagen binding domain. The aim of this study was to identify the binding ligand of CbpA and the function of CbpA in cellular processes that are part of the B. cereus group c-di-GMP regulatory network. By global gene expression profiling cbpA was found to be down-regulated in a cdgF deletion mutant, and cbpA exhibited maximum expression in early exponential growth. Contrary to the wild type, a ΔcbpA deletion mutant showed no binding to collagen in a cell adhesion assay, while a CbpA overexpression strain exhibited slightly increased collagen binding compared to the control. For both fibrinogen and fibronectin there was however no change in binding activity compared to controls, and CbpA did not appear to contribute to binding to abiotic surfaces . Also, the CbpA overexpression strain appeared to be less motile and showed a decrease in biofilm formation compared to the control. This study provides the first experimental proof that the binding ligand of the c-di-GMP regulated adhesin CbpA is collagen.Cyclic diguanylate (c-di-GMP) signalling affects several cellular processes in  Biofilmix genes . Most B.ectively . One of x status .cdgF deletion mutant (B. thuringiensis 407 (cry-) which is under c-di-GMP transcriptional control and linked to CdgF function . Their genomes and proteomes were searched with the cbpA/CbpA sequence from B. thuringiensis 407 using BlastN and BlastP. InterProScan (http://service.iiserkol.ac.in/~riboscan/application.html) was used to explore DNA sequences for class I c-di-GMP riboswitches.At the time of analysis 144 fully sequenced and closed genomes of members of the rProScan  was utilrProScan . The webrProScan  (http://2.2B. cereus group. The tree was constructed as in http://mlstoslo.uio.no). Tree construction and visualization was executed using Seaview 4 (Altogether 113 strains were used to build a phylogenetic tree representative of the eaview 4  and Treeeaview 4 .2.3B. thuringiensis strain 407 Cry- (600) of 0.05 from an overnight culture and grown at 30 ͦC and 220 rpm in Luria Bertani (LB) broth or bactopeptone medium . For cloning and expression in Escherichia coli, ampicillin was used at 50 or 100 µg ml−1, kanamycin at 50 µg ml−1, erythromycin at 200 µg ml−1 (pMAD), and tetracycline at 12 µg ml−1. Erythromycin was used at 10 µg ml−1 to maintain the pHT304-Pxyl plasmid constructs in B. thuringiensis. Unless otherwise indicated, induction of gene expression from the xylA promoter in pHT304-Pxyl was performed by addition of 1 mM xylose to the growth medium.The model strain used in this study is the acrystalliferous 407 Cry- . Culture2.4E. coli/Bacillus shuttle vector pHT304-Pxyl was used, in which xylR and the xylA promoter from B. subtilis was inserted into the pHT304 cloning site  values. The expression of the target gene in each biological replicate was converted into ECq values  and was precipitated (20 μg) in 0.3 M NaAc (pH 5.5) and 70% ethanol overnight at −80 °C ahead of cDNA preparation. Microarray slides were printed at The microarray core facility of the Norwegian University of Science and Technology (NTNU). Design, printing, prehybridization, hybridization and scanning of the slides, and analysis of the data, was performed as described in RNA isolation, cDNA synthesis, labeling and purification was performed as previously described . RNA wasB. anthracis Ames, B. anthracis A2012, and B. cereus ATCC 14579, in addition to selected genes from B. cereus ATCC 10987 , and the gene lists used for microarray analysis are based on the annotations from this GenBank entry. Only probes with 93% identity or greater to a transcript/feature sequence of B. thuringiensis 407 were included in the analysis. COG categories were obtained for the analyzed genes as reported in the IMG database (http://img.jgi.doe.gov). The data obtained from this analysis was submitted to the ArrayExpress archive (https://www.ebi.ac.uk/arrayexpress/) under the accession number E-MTAB-8092.The microarray slides contain 70-mer oligonucleotide probes designed to detect open reading frames (ORFs) in CC 10987 . To faci2.7600 ≈ 0.3) were used to inoculate fresh bactopeptone medium to an OD600 of 0.01. For each strain, four wells of a 24-well polystyrene plate  were filled with 500 µl of the bacterial culture suspension. Plates were produced in duplicate, and each plate contained four wells of bactopeptone medium as control. Following incubation at 30 ͦC for 24 h, 48 h and 72 h, the wells of one of the microplates were washed once with phosphate-buffered saline (PBS) and stained with an 0.1% (w/v) aqueous solution of methyl violet 6B for 30 min at room temperature. The wells were then washed three times with PBS and dried upside down over night. For quantification of biofilm formation, the dye was solubilized by adding 500 µl of a 1:4 acetone/ethanol mixture to each well and incubated for 10 min at room temperature, followed by measuring the absorbance at 575 nm in a plate reader (BMG Labtech ClarioStar).Biofilm forming capability was investigated in a multi-well plate screening assay, modified from a previously described method . Culture2.8600 ≈ 1.0, and 5 µl of culture was spotted on LB agar plates containing 0.3% agar, with 1 mM xylose and/or 10 µg ml−1 erythromycin added if appropriate. Plates were incubated for 7 h at 30 °C and motility was measured as the distance between the colony edge and the outer line of the swimming zone.Motility was assessed essentially as described in 2.9−1 in 0.01 M acetic acid, while fibrinogen and fibronectin were diluted in PBS. 96-well polystyrene microplates (Corning 3370) were coated with 125 µl per well of each substance for a duration of 1 h at room temperature. After blocking with 50 mg ml−1 bovine serum albumin  in PBS for 30 min at room temperature, the wells were washed three times with 0.1 mg ml−1 BSA in PBS. Ahead of the assay, bacteria were grown to early exponential phase at 30 ͦC (OD600 ≈ 0.2–0.3) in LB medium, supplemented with 10 µg ml−1 erythromycin and 1 mM xylose for strains containing the pHT304-Pxyl plasmid. Bacterial culture suspensions were centrifuged for 15 min at 1500×g, resuspended in PBS and adjusted to an OD600 of 0.9–1.0. For each strain in each independent experiment, eight collagen-coated wells and eight empty wells, representing technical replicates, were filled with 125 µl of the bacterial suspension and incubated for 1 h at room temperature. Additionally, eight collagen-coated and eight empty wells  were incubated with PBS as negative controls. After washing three times with PBS, wells were stained with 125 µl of an 0.1% (w/v) aqueous solution of methyl violet 6B for 30 min at room temperature. Solubilization of the dye was performed as described for the biofilm assay, but with 125 µl 1:4 acetone/ethanol mixture. Absorbance at 575 nm was measured in a plate reader (BMG Labtech ClarioStar).Binding to biotic surfaces was investigated with a whole cell adhesion assay adapted from the method described in 2.102O and incubation in acetone for 15 min at room temperature. After four subsequent washes in dH2O the steel coupons were autoclaved. Glass cover slips were autoclaved and all plates were dried overnight. The adhesion assay is based on a method described by Hayrapetyan and co-workers . Steel coupons were prepared as described earlier  by treat-workers . Coupons2.11B. thuringiensis 407 wild type (Bt407), the cbpA deletion strain (Bt407ΔcbpA), and the complementation strain (Bt407ΔcbpA pHT304-Pxyl-cbpA), by infection (force feeding) in 5th instar Galleria mellonella larvae. G. mellonella eggs were hatched at 28 °C and the larvae reared on beeswax and pollen. For infection experiments, groups of 20 to 25 G. mellonella larvae, weighing about 200 mg were used. Xylose (20 mM) was added to the LB growth medium of all strains, as well as to the bacterial inoculums and the toxin alone control (Cry1C) at time zero (time point of force feeding). Larvae were force fed a second time with 10 µl 20 mM xylose 5 h later (in order to again activate CbpA expression from the pHT304-Pxyl plasmid in the complementation strain). Infections were otherwise performed as previously described  and 3 µg of activated Cry1C toxin. The larvae in the control group were fed either 10 μl PBS buffer or 10 μl Cry1C toxin + xylose. Mortality of the infected larvae was observed after 4 h, 24 h and 48 h. The chosen dose was expected to result in about 70 (±5) % larvae mortality for the infection with the wild type B. thuringiensis 407 at 37 °C after 48 h -adjusted p-value < 0.05; cdgF deletion mutant, suggesting that this gene was directly or indirectly induced by CdgF. Expression of the two other genes, one encoding a putative aldo/keto reductase and the other a putative MFS-type transporter, was upregulated 2.5-fold  and is annotated as a pseudogene due to a frameshift mutation in the closed B. thuringiensis 407 genome  promoter region showed that it contains a match to a class I c-di-GMP responsive riboswitch (GEMM), originally identified and named Bc2 in B. cereus ATCC 14579 by Sudarsan and co-workers (cdgF deletion mutant mediates regulation of expression of BTB_RS05575 (BTB_c11270).BTB_RS05575 (BTB_c11270) was interrupted by a gap between two contigs in the first available draft genome sequence of 18877.1; ), potent18877.1;  matching-workers . This ri-workers , suggestB. thuringiensis 407 is predicted to be 2187 amino acids in length, and contains an N-terminal signal peptide and a C-terminal cell wall sorting signal comprising an LPXTG sortase substrate motif (Streptococcus pneumoniae (Staphylococcus epidermidis (S. aureus ClfA (S. aureus SdrD (S. aureus (S. aureus collagen binding MSCRAMM protein Cna (S. aureus Cna (The corrected BTB_RS05575 (BTB_c11270) coding sequence in te motif . The prote motif  further te motif . The noneumoniae  . cbpA was not identified in B. anthracis or any emetic-toxin producing strains which group within phylogenetic cluster III, and only four of the 40 B. cereus or B. thuringiensis strains belonging to cluster III contained cbpA.Since boswitch  and the us group , we inveV and VI . The surV and VI  and the pipeline  and no ipipeline . Phyloge insects , http://cbpA gene, but based on the genome sequences present in GenBank carry genetic changes implying the strains may carry cbpA pseudogenes or alternatively, as found in our resequencing of B. thuringiensis 407 cbpA, may be the result of genome sequencing errors due to extensive internal repeat regions in the cbpA gene. All strains that contained the cbpA gene (or pseudogene) also contained a predicted class I c-di-GMP binding riboswitch in the upstream untranslated region (UTR).The highest variability within CbpA of the analysed strains was observed in the B-region and in the proline-rich region , while t3.5cbpA and corresponding cbpA complementation- and overexpression strains utilizing the xylose-inducible vector pHT304-Pxyl were constructed , binding was completely abolished to all types of collagen tested, while the cbpA overexpression strain (Bt407 pHT304-Pxyl-cbpA) showed statistically significant increased adhesion to collagens I – IV compared to the vector control strain (Bt407 pHT304-Pxyl) (cbpA pHT304-Pxyl-cbpA) to roughly the same levels as in the wild type strain for all tested collagens . The adhesion assay clearly revealed that under the tested conditions, CbpA is essential for binding to collagen.To uncover a possible host cell molecule binding to CbpA, a markerless gene deletion mutant Δstructed . The capth phase A bacteri04-Pxyl) A. As a ccbpA did however not produce any difference in adhesion to either fibrinogen or fibronectin compared to B. thuringiensis 407 wild type. In fact, under the tested conditions none of the strains produced detectable binding of the bacterial cells to wells coated with either of these two molecules  formed less biofilm compared to the vector control strain  had a significantly smaller swimming zone compared to the vector control strain (Bt407 pHT304-Pxyl), indicating an impairment in motility upon cbpA overexpression (600 = 0.2) and stationary phase, obtaining similar results compared to the original assay  compared to the wild type.Since adhesion to host factors is the first step of infection, the function of MSCRAMMs is usually predicted to be connected to infection processes . In ordea larvae . We did 4B. thuringiensis MSCRAMM protein CbpA mediates binding to collagen, an extracellular host cell matrix protein. The CbpA ortholog in the B. thuringiensis strain BMB171 has been subject of a study by Tang and colleagues (cbpA deletion mutant. In our study, CbpA is essential for B. thuringiensis 407 attachment to collagens I, II, III, IV and V, for bacterial cells in exponential growth phase. Results from the adhesion assays further suggest that CbpA is not able to bind to neither fibrinogen nor fibronectin, contrary to several other MCSRAMM proteins (B. cereus group cap genes are a hallmark for genes responsible for synthesis of the poly-γ-D-glutamic acid capsule (To the best of our knowledge this study presents the first experimental evidence that the lleagues , which fproteins . We prop capsule , typicalB. cereus group continue to pose a significant problem in the food industry, and the ability of these bacteria to bind to abiotic surfaces, especially steel which is commonly used in food processing plants, has been investigated previously (Members of the eviously . Althougeviously , as welleviously , there icbpA expression is regulated at least partly by an upstream c-di-GMP binding riboswitch (B. thuringiensis 407 (cbpA is positively regulated by c-di-GMP, it could be expected that cbpA expression in biofilm cells would be high (cbpA expression was not at its peak in biofilm cells, but in planktonic cells during the early exponential growth phase, possibly indicating that expression of cbpA is subject not only to transcriptional control by c-di-GMP, but potentially also by other mechanisms. We did not detect a significant difference in biofilm mass nor in motility upon cbpA deletion in B. thuringiensis 407. In contrast, Tang and colleagues (cbpA deletion mutant of B. thuringiensis BMB171. This is surprising, since B. thuringiensis 407 and B. thuringiensis BMB171 are closely related strains within the B. cereus group, and the sequence identity between the predicted binding domains N1-N3 of CbpA in these two strains is 97%. Although we could not relate the absence of CbpA from the B. thuringiensis 407 cell surface to biofilm or motility phenotypes, the overexpression of CbpA in B. thuringiensis 407 led to a decrease in both biofilm formation and motility. This is in contrast to the common pattern of c-di-GMP regulatory networks where motility and biofilm formation are normally oppositely regulated (B. thuringiensis 407 (Biofilm formation and motility are cellular processes which are typically affected by c-di-GMP signalling . Since cboswitch , we aimensis 407  and that be high . Analysilleagues  report begulated , and whinsis 407 , possiblnsis 407 . With thB. cereus are gastrointestinal tract infections, but the bacterium is also known to cause a variety of other infections, particularly in immunocompromised patients (B. cereus group strains isolated from non-clinical sources. Interestingly however, all collagens that were used in the adhesion assay in this study originate from mammalian tissues, providing a potential for CbpA to make a contribution to attachment of B. cereus group strains to tissues also during human infection. Also, while B. thuringiensis is often considered non-pathogenic for humans and is widely used as an biological pesticide (B. thuringiensis strains generally contain the same chromosomal virulence factors that are often associated with human infections caused by B. cereus. This includes the Hbl, Nhe and CytK cytotoxins (B. cereus group infections is not completely elucidated. Yet these proteins are generally theorized to cause food-borne diarrhoeal disease by disruption of the epithelial layer, functioning as enterotoxins. Also, the insecticidal Cry toxins of B. thuringiensis function by forming pores in the epithelial cells in the insect midgut (in vivo infection assay in the wax moth G. mellonella larvae did not reveal a significant decrease in mortality for the cbpA deletion strain. Quite possibly, this could be explained by genetic redundancy, as B. thuringiensis 407 contains two other proteins that are annotated as collagen adhesins, as well as an ortholog to B. anthracis BA0871 for which binding of collagen has been shown experimentally (B. thuringiensis, the protein could still facilitate adhesion during the colonisation of a host and it is interesting to observe that a slight trend towards increased virulence was observed when the cbpA gene is expressed from the low copy plasmid pHT304-Pxyl in the complementation strain. Also, B. thuringiensis has been suggested to have the capacity to form biofilm in the insect gut (reviewed in B. thuringiensis strain BMB171, although carrying a similar complement of putative collagen adhesion proteins to B. thuringiensis 407, indeed affected virulence towards the insect larvae Helicoverpa armigera. Here the in vivo assay was however run under substantially different experimental conditions, using young larvae and bacteria in the sporulation stage, and the effect measured over  six days following free feeding. This is different from the G. mellonella analysis performed in the present study, with a one shot dose of a controlled number of vegetative bacteria on large larvae, possibly explaining the difference in obvious impact.Most infections caused by patients . Collagepatients , and colpatients . Collagepatients . The phyesticide , some stesticide  and B. ttotoxins , the funt midgut . One maymentally  (Xu et amentally . AlthougB. cereus group strains, and may constitute a novel surface protein potentially involved in adhesion processes during infection.This study presents the first experimental proof that the extracellular matrix protein collagen is a ligand of the CbpA cell surface adhesin, and that CbpA-mediated adhesion is collagen-specific and not implicated in adhesion to fibrinogen or fibronectin, nor to tested abiotic surfaces. CbpA is present in a range of The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Low health literacy is believed to be associated with behaviours that increase the risk of type 2 diabetes. But there is limited knowledge on the relation between health literacy (HL) and diabetes risk, and whether improving HL could be a potential prevention strategy. Therefore, the main purpose of this study was to examine the link between HL and diabetes risk among non-diabetic adults.We used data from the Hamburg Diabetes Prevention Survey, a population-based cross-sectional study in Germany. One thousand, two hundred and fifty-five non-diabetic subjects aged 18–60 years were eligible. The German Diabetes Risk Score  was used to determine the individual risk of type 2 diabetes. The short version of the European Health Literacy Questionnaire  was applied to assess the individual self-reported HL. Subjects were asked to self-estimate their diabetes risk, which was then compared with the calculated GDRS. Descriptive statistics were calculated to investigate group differences in the GDRS and self-estimated diabetes risk. Linear as well as logistic regression models were performed to analyse potential influencing variables of the GDRS as well as incorrect self-estimated diabetes risk. In three nested statistical models for each outcome, these analyses were adjusted for age, gender, educational level and the presence of chronic conditions.P = 0.020) and had a 2.04 greater chance to estimate their diabetes risk incorrectly  compared to those with “sufficient HL”.According to the criteria of the GDRS, 996 (79.4%) subjects showed “low risk”, 176 (14.0%) “still low risk”, 53 (4.2%) “elevated risk”, and 30 (2.4%) “high to very high risk” to develop type 2 diabetes within the next 5 years. In the statistical models including all control variables, subjects with “inadequate HL” scored 2.38 points higher on the GDRS (95% CI 0.378 to 4.336; The risk of type 2 diabetes is increased in people with inadequate self-reported HL. People with high diabetes risk and inadequate HL might be provided with educational programs to improve diabetes knowledge and reduce behavioural risk factors.The online version contains supplementary material available at 10.1186/s12889-021-10508-2. About 425 million adults worldwide are estimated to have diabetes mellitus, causing total healthcare expenditures of 727 billion USD yearly . Around . Low HL is associated with behaviours that increase the risk of type 2 diabetes such as smoking, inactive lifestyle and poor dietary habits [Due to urbanization and sedentary lifestyle, the prevalence of type 2 diabetes is rising worldwide , and newy habits . Prior fy habits , 7, and y habits , 8–10. Ry habits , 12.However, in Europe, every second adult has limited HL  and therFor this analysis we used data from the Hamburg Diabetes Prevention Survey (HDPS), a population-based cross-sectional study in Germany. The survey was conducted between December 2017 and March 2018 using computer-assisted telephone interviews.For the HDPS, a sample of private households in Hamburg was selected by randomly dialling landline phone numbers. The selection was made according to the selection framework for telephone samples from Arbeitskreis Deutscher Markt- und Sozialforschungsinstitut (ADM) , a businA minimum sample size of 1000 randomly selected participants was defined in the study. To better represent the low socioeconomic groups, two additional waves of data collection were conducted, in which 1) a minimum of 150 subjects with no school certificate or low secondary education and 2) a minimum of 150 subjects with low or intermediate secondary education were recruited. As the HDPS was an observational study with multiple outcomes, it was not possible to carry out a sample size calculation. Instead, the sample size was determined based on the principal investigators’ experience with similar studies.. If the subjects from the additional waves did not meet the inclusion criteria 1) inadequately completed general education, general elementary education or basic vocational qualification or 2) intermediate qualification or A level equivalent, but no tertiary education, the interview was ended after the screening questions and the subjects were excluded from the study.The population was selected by two screening questions at the beginning of the interviews The survey based upon a standardised questionnaire including validated instruments of the 8-item Short-Form Health Survey (SF8) to measure health-related quality of life , the shoFor the HDPS, individuals 18–60 years old who answered the phone, and spoke German were included in the sample  classification . AccordiSelf-reported HL was assessed using the HLS-EU-Q16 , 23, whiDependent variables included a clinical assessment of diabetes risk and the self-estimated diabetes risk.The clinical assessment of the risk of type 2 diabetes was assessed using the German Diabetes Risk Score (GDRS) , 29. TheWe categorised the GDRS (ranging 0 to 123 points) according to the recommendations of the DIfE as follows: < 46 points for “low risk” (< 2% will develop type 2 diabetes within the next 5 years), 46–56 points for “still low risk” (2 to 5%), 57–63 points for “elevated risk” (6 to 10%) and > 63 points for “high to very high risk” (> 10%) , 30. Our“Compared to other people of my age and gender, I have a lower risk of developing diabetes”. The responses “strongly agree” and “agree” were dichotomized to “yes”, the responses “disagree” and “strongly disagree” were dichotomized to “no”, and the response “I don’t know” was treated as missing value. To examine if subjects were able to estimate their diabetes risk correctly, the responses were compared with the diabetes risks resulting from the GDRS.To assess the self-estimated diabetes risk, subjects were asked to agree or disagree to the following question on a four point Likert scale: A correct estimation of the own diabetes risk was assumed if they answered “yes” and had a “low” or “still low” risk according to the GDRS or if they answered “no” and had a “elevated” or “high to very high” risk according to the GDRS. An underestimation was assumed if they answered “yes” and had an “elevated” or “high to very high risk” according to the GDRS. An overestimation was assumed if they answered “no” and had a “low” or “still low” risk according to the GDRS. For the analyses, underestimating or overestimating of own diabetes risk was considered as incorrect estimation. Self-estimated diabetes risk was assessed for all subjects with available data regarding these items.We calculated descriptive statistics for socio-demographic variables and investigated group differences in the GDRS and self-estimated diabetes risk based on the categories described above. We analysed the association between health literacy (independent variable) and the GDRS (dependent variable) by a multivariate linear regression analyses. Before analysis, we used a kernel density plot to assess normality for the diabetes risk score, which showed a slightly skewed, but approximately normal distribution for this variable  and odds ratios (OR) were reported indicating 95% confidence interval (CI). Additionally, we assessed if the interaction between health literacy and educational level had a significant association with 1) clinical diabetes risk and 2) incorrectly self-estimated diabetes risk. The level of statistical significance was set at ionnaire .Initially, 1147 subjects were interviewed for the HDPS. To better represent the low socioeconomic groups, additional 150 subjects with no school certificate or low secondary education as well as 158 subjects with low or intermediate secondary education were interviewed. The HDPS sample included 1455 subjects in total.For our study, we used the HDPS sample, but excluded 75 subjects who reported diagnosis of diabetes mellitus, and excluded 125 subjects due to missing values. Finally, 1255 subjects were eligible  consisted of subjects aged 50–60 years, while subjects aged 18–29 years constituted the smallest age group  (Table 576 (45.9%) subjects were male and the mean age was 43.1 years (SD: 12.0). The largest age group  to 55.6%   subjects showed “inadequate”, 414 (33.0%) “problematic” and 708 (56.4%) “sufficient” HL (Table 1) Table . The CroAccording to the criteria of the German Diabetes Risk Score (GDRS), 996 (79.4%) subjects had “low risk” (5 years disease probability < 2%), 176 (14.0%) “still low risk” (2 to 5%), 53 (4.2%) “elevated risk” (6 to 10%), and 30 (2.4%) “high to very high risk” (> 10%) to develop type 2 diabetes within the next 5 years (Table n = 24) in the age group 50–60 years compared to the age groups 40–49 , 30–39  or 18–29 . In all three models, increasing age was associated (P < 0.001) with higher risk of type 2 diabetes (Table The diabetes risk differed between age groups Table . The ratn = 22) as well as “high to very high diabetes risk”  was lower than in male groups . Female gender was consistently associated (P < 0.001) with lower risk for type 2 diabetes  higher than in the medium  and high education group . In both respective models, education was inversely associated with the risk of type 2 diabetes (Table P < 0.001). A 2.276 points decrease in the score was seen for medium  compared to low education  as compared to study participations reporting not to be chronically ill . People suffering from chronic diseases had a 4.041 (P < 0.001) point higher GDRS than people without chronic diseases. Including education and the presence of chronic conditions into our statistical models did in both cases improve the model fit (both P < 0.001).Table n = 23) in groups with “problematic” HL by 5.3% (n = 22) and in groups with “inadequate” HL by 6.1% (n = 8) (Table n = 6) was in the “inadequate” HL group the highest, compared to the groups with “problematic”  or “sufficient” HL . However, in across all three multivariate statistical models, “problematic” HL did not significantly affect the GDRS (P = 0.419). In contrast, “inadequate” HL was significantly associated with a 2.38 points higher German Diabetes Risk Score  compared to “sufficient” HL.We also found differences in diabetes risk by HL level Fig. . The ratp = 0.686) and inadequate HL  and medium educational level and b) problematic  and inadequate HL  and high educational level with the GDRS, therefore interactions were not included in the final models in Table We did not find an association of a) problematic  were excluded from the analyses due to missing values. Of 1107 subjects (88.2%) with self-estimated diabetes risk, 751 (67.8%) estimated their risk correctly. Three hundred fifty-six subjects (32.2%) underestimated or overestimated their diabetes risk (Table P = 0.016). In all three multivariate models, we found no association (P = 0.075) between incorrectly self-estimated diabetes risk and gender , 35.5% (n = 195), and 38.0% (n = 68) in groups with high, medium and low education, respectively. In our multivariate models, medium education was consistently associated with a higher chance of incorrectly self-estimated diabetes risk  compared to high education (Table P = 0.042) in model 3, but slightly larger if not controlled for chronic conditions .We could see differences in self-estimated diabetes risk by educational level Table . The rat8.0% n = 8 in groun = 162 vs 27.0%, n = 191) (Table P < 0.001) (Table P = 0.002) and the presence of chronic conditions (P > 0.001) into our statistical models.Study participants reporting to be chronically ill had a higher risk for incorrectly self-estimated diabetes risk, both in descriptive analyses  Table  and in on = 174) in groups with “sufficient” HL, 36.0% (n = 130) in groups with “problematic” HL and 46.8% (n = 52) in groups with “inadequate” HL, respectively  compared to “sufficient” HL (Table P = 0.001).In all three multivariate models, “problematic” HL was associated with a higher chance of incorrectly self-estimated diabetes risk  and inadequate HL  and medium educational level and b) problematic  and inadequate HL  and high educational level with the incorrectly self-estimated diabetes risk. Therefore, for this outcome, there were also no interactions included in the final model in Table There was no association of the interaction between a) problematic  and the risk of type 2 diabetes among non-diabetic adults. Our results demonstrated that the risk of type 2 diabetes is increased in people with inadequate HL, but not in subjects with problematic HL. We found that adults with problematic and inadequate HL tend to underestimate or overestimate their diabetes risk significantly. For both outcomes, we found no interaction between HL and the educational level.Compared to a representative survey among users of the statutory health insurance in Germany , the ratIncreasing age is a non-modifiable risk factor for type 2 diabetes . In lineWe observed a lower risk of type 2 diabetes for women than for men. Results from six population-based studies in Germany demonstrated, that diabetes prevalence was significantly lower among women than among men . MoreoveIn our study, chronically ill subjects had a higher diabetes risk and also a higher probability for an incorrectly self-estimated diabetes risk than people who did not report to be chronically ill. A possible explanation for this finding is that many chronic diseases are either risk factors for diabetes or share the same behavioural or genetic risk factors with diabetes (e.g. cardiovascular diseases or gout) –39.n = 42) using the Finnish Diabetes Risk Score (FINDRISC) [n = 1279) using the Australian Type 2 Diabetes Risk Assessment Tool [In our study, lower educational level was associated with higher diabetes risk among non-diabetic adults. A prevalence study from Germany revealed that the risk of having diabetes was significantly higher for low educational level compared with medium or high levels . FurtherINDRISC)  and the ent Tool . To our . Two of them suggested that lower HL was associated with higher diabetes prevalence [. One was based on data of older US Americans [, the representative survey among users of the statutory health insurance in Germany did not identify such an association [However, there are previous studies that analysed the link between HL and diabetes prevalence , 7, 26. ociation , 10. Sarociation . SchilliOur results also demonstrated that non-diabetic adults with inadequate HL had a significantly higher chance to estimate their diabetes risk incorrectly, compared to adults with adequate HL. One possible explanation might be that people with inadequate HL know less about chronic diseases than those with adequate HL as shown by other studies , 43, 44.Overall, HL seems to have an impact on health related outcomes such as diabetes. According to the World Health Organization (WHO), HL is an important factor in preventing type 2 diabetes, as low HL is associated with modifiable risk factors such as smoking, poor diet and physical inactivity . The GerIn line with findings from previous publications, our results suggested a higher diabetes risk for men , 36, 45,One strength of our study was the use of the German Diabetes Risk Score (GDRS) , 30–33 iComparing our results with findings from other studies could be difficult, because in recent studies two versions of HLS-EU (Q16 and Q47) were applied and differently evaluated to assess HL , 23–26. Our analyses were based on a cross-sectional survey, which shows the correlation between HL and the risk of type 2 diabetes, but gives no information about the change over time and the direction of this association, e.g. if risk factors for diabetes also affect the patients’ health literacy or if health literacy has an influence on the diabetes risk. Our results should therefore be interpreted cautiously. Our data were collected using telephone interviews therefore socially-desirable answers and selection bias could be existent. The interviews were conducted in German language only. Non-German speaking adults were not interviewed which could have led to overestimation of HL in the study population , 46. AlsAdditionally, the HDPS has a slightly uneven distribution of women (54.1%) and men (45.9%) in the sample, which probably affects the representativeness of the descriptive data. However, our multivariate data analyses identifying predictors of GDRS and incorrectly self-estimated diabetes risk have been adjusted for gender. Therefore these results should still be valid for the selected age groups in the study area.n = 6272) that completed the telephone interview. However, it has to be taken into account, that in our approach of generating randomized telephone numbers, many numbers presented a free line signal, but did not belong to a household. Considering this fact, our response rate would have been much higher. Furthermore increasing age is strongly associated with type 2 diabetes, thus it is most commonly seen in elderly people [Another limitation is the low rate of participants  and the short version of the European Health Literacy Questionnaire (HLS-EU-Q16) are practicable tools to select people at high diabetes risk that have inadequate health literacy. In order to prevent type 2 diabetes or to delay its onset, this subgroup might be provided with educational programs improving their diabetes knowledge and supporting them to change their lifestyle. Future prospective-controlled trials are needed to confirm this assumption.Additional file: Supplementary File 1. Kernel density estimate. It shows a slightly skewed, but approximately normal distribution for the diabetes risk score.


Due to a production error, several words were incorrectly capitalized throughout the text. These have now been correctly capitalized.Equations 17–20, there were several instances where the authors mistakenly switched the positive and negative signs.In Statistical Significance and Simulation of the Models section, subsection Recognition Accuracy and Comparison and End-to-End Deep Learning Architectures, paragraph 12, the authors accidently missed the addition of a footnote. The following footnote has been added for UCF-101: https://www.crcv.ucf.edu/data/UCF101.php.In the Table 7, “Kingma and Ba (2014)” should be replaced with “Tran et al. (2015).” Following that, “Tran et al. (2015)” should be replaced with “Competé et al. (2019).”In Figure 6, a sentence has been added to clarify that this is based on “G2,4” where 2 & 4 are pointers.In the legend for Statistical Significance and Simulation of the Models section, subsection Forecasting Ability for Motion Trajectories, paragraph 2, The final two mentions of “G2,1” have been corrected to “G2,4.”In the The publisher and authors apologize for these errors. The original version of this article has been updated.


Pkhd1 gene cause autosomal recessive polycystic kidney disease (ARPKD). Pkhd1 encodes fibrocystin/polyductin (FPC), a ciliary type I membrane protein of largely unknown function, suggested to affect adhesion signaling of cells. Contributions of epithelial cell adhesion and contractility to the disease process are elusive. Here, we link loss of FPC to defective epithelial morphogenesis in 3D cell culture and altered cell contact formation. We study Pkhd1-silenced Madin-Darby Canine Kidney II (MDCKII) cells using an epithelial morphogenesis assay based on micropatterned glass coverslips. The assay allows analysis of cell adhesion, polarity and lumen formation of epithelial spheroids. Pkhd1 silencing critically affects the initial phase of the morphogenesis assay, leading to a reduction of correctly polarized spheroids by two thirds. Defects are characterized by altered cell adhesion and centrosome positioning of FPC-deficient cells in their 1-/2-cell stages. When myosin II inhibitor is applied to reduce cellular tension during the critical early phase of the assay, Pkhd1 silencing no longer inhibits formation of correctly polarized epithelia. We propose that altered sensing and cell interaction of FPC-deficient epithelial cells promote progressive epithelial defects in ARPKD.Mutations of the  Ciliopathies are rare (mostly) monogenetic diseases (or syndromes) caused by mutation i.e., loss of function, of proteins of the cilia-centrosome complex . The ranThroughout the bodies of multicellular organisms, epithelia form barriers that separate tissues from liquid- or gas-filled lumina. Epithelial function depends on establishment and maintenance of defined apicobasal polarity and control of cell(–cell) interactions in the epithelial plane that distribute forces and define permeability of the barrier ,10,11. TIn brief, the (i) distribution of cellular contractile elements (cell contractility) through connection of the actin cytoskeleton to different receptors and the (ii) positioning of centrosomes, the organization centers of microtubules (MTOCs), are pivotal to the function of epithelial sheets and influence maintenance of mechanical stability, force distribution and permeability of the barrier. Cellular control of both essential features can be assessed during epithelial cell spheroid formation. For Madin-Darby Canine Kidney II (MDCKII) cells, Rodríguez-Fraticelli and colleagues  developeHere, we address consequences of a transient reduction of FPC protein on the capacity of collecting duct epithelial cells (MDCKII cells) to form correctly polarized, monolayered spheroids, i.e., to control initial steps of epithelial morphogenesis. The assay starting from a single cell specifically probes whether cell(–cell) interactions and balance of forces are altered in cells deficient of FPC function. In these conditions, MDCKII cells with insufficient FPC expression display a strongly compromised capacity to form correctly polarized epithelia, a result of altered behavior in their one-cell and two-cell stages. Upon treatment to reduce contractile forces during the critical, initial phase of spheroid formation, control of epithelial polarity is restored, confirming deficits of FPC-deficient cells in cellular control of cytoskeletal forces and adhesion signaling. Altered sensing and interaction of epithelial cells with their environment are suggested to lead to progressive defects in collecting and bile duct epithelia and eventually loss of organ function in ARPKD patients.Epithelial morphogenesis by MDCKII cells is highly sensitive to adhesion signaling. In cell culture, loss of FPC (function) from epithelial cells was proposed to associate with altered adhesion signaling and thus changes in environmental sensing and cell interactions [+/K+ ATPase) markers [ractions . To addrractions . Imposin markers ,16 and (2 (high cell confinement) provides a balanced mix of stimuli that induce formation of correct spheroids (groups 1 and 2) with high incidence of greater than 85%, 2 (low cell confinement), which, due to their larger adhesive area do not mimic the spatial constraints imposed on cells in epithelial monolayers, generate 43% of groups 1 and 2 spheroids and more than 50% of cell aggregates. Furthermore, adhesion of cells to laminin, which induces only weak cell–ECM interactions, allows efficient formation of correct spheroids (groups 1 and 2) independent of the confinement imposed by disc-shaped patterns (700 or 1600 µm2). These data confirm that the spheroid assay is functional and operates in agreement with observations by Rodriguez-Fraticelli et al. [Function of the assay was confirmed as detailed in i et al. . Thus, iTemporal loss of FPC function leads to defective spheroid formation. Impaired FPC function is achieved by transient knockdown of Pkhd1 expression using specific siRNA (siPkhd1) or shRNA (shPkhd1) as validated and reported for MDCK cells by Zhang and colleagues [Pkhd1 mRNA levels down to 15–30% of control treatment. The reduction is observed 48 h after siRNA treatment and 48–72 h after induction of shRNA expression using doxycycline. We control effectiveness of siRNA/shRNA treatment using quantitative real-time PCR to determine mRNA levels of Pkhd1 and three reference genes. Knockdown is similarly effective when using siPkhd1, shPkhd1 or the pool of four Pkhd1-specific shRNAs (shPoolPkhd1) that were described previously [Pkhd1 silencing was reported to alter PC2 expression [Pkhd1 silencing, we also detected loss of the full-length FPC protein with an apparent molecular weight of 450 kDa in cells expressing shPkhd1 and shPoolPkhd1 with and without doxycycline induction (Pkhd1 silencing are underestimated due to approximately 20% of cells with no (or insufficient) effect of treatment.lleagues . Consistpression . Therefonduction D. StrongPkhd1 treatment is timed to coincide maximal knockdown of FPC with cell seeding, one-cell and two-cell stages, while mRNA regains wildtype levels, when spheroids are ready for classification after three days  in siPkhd1 compared to siControl-treated cells (Pkhd1 (or shPoolPkhd1)  . DefectiFPC-deficient cells show altered control of spreading and adhesion. Results of the spheroid assay suggest a significant impact of FPC protein function on the initial steps of spheroid formation and by implication epithelial morphogenesis. Does this correspond to an altered control of cell shape and adhesion? To study this possibility, we collected quantitative adhesion parameters of MDCKII cells 4 h after seeding onto 700 or 1600 µm2 collagen-coated micropatterns, employing identical culture conditions as in the spheroid assay. After 4 h, cells are fully spread and adapted to cell-ECM adhesion (steady-state). Detection of FAs, by fluorescence staining of cytoskeletal adaptor proteins such as vinculin, allows extraction of number, size and area of adhesion sites, a semi-quantitative estimate of adhesion strength [Pkhd1 and siControl-treated cells on both sizes of collagen-coated micropatterns (700/1600 µm2) are shown in 2, the shape of siControl cells reflects adaptation to confinement by completely filling the available area, while adhesion to 1600 µm2 induces an elongated, polarized cell shape. This is observed also for both conditions by the circularity index measuring the ratio of the length axis to the perpendicular width of cells , whereas cells are more than 50% longer than wide with low lateral restriction  than siControl cells on 700 µm2, rarely filling the entire pattern, and show a moderately elongated shape (circularity of 0.78)  is not altered, compared to siControl cells. In addition, release of confinement triggers a significant increase in cell spreading area (14.6%) and number of cell adhesions (26.5%), indicating that confinement also affects the control of adhesion sites in siPkhd1 cells  A–C. To aferences . Since sd1 cells C–E. TherCentrosome positioning is affected in one-cell and two-cell states of FPC-deficient cells. Relative orientation of organelles reflects the ability of cells to correctly read and respond to environmental cues [2 crossbow-shaped micropatterns is optimal for MDCKII cells, leading to no detectable differences in shape (aspect ratio) or size of siPkhd1 cells and controls -inhibitor RO-3306, 9 µM) and determined positions of centrosomes and nuclei in their two-cell stages 6 h after inhibitor washout. Cells without siRNA treatment were studied in control experiments to confirm centrosome positioning on 700 µm2 as compared to 1600 µm2 patterns. 2; for measurement, see 2, reduced FPC expression also leads to a detectable and significant reduction of the nuclear to centrosomal distance ratio by 12% of siPkhd1 compared to siControl-treated cells.Initiation of lumen in epithelial spheroids is accompanied by positioning of centrosomes relative to nuclei, which is also an indicator of force distribution between adjacent cells pairs . In highTimed reduction of actomyosin contractility rescues spheroid formation of FPC-deficient cells. As observed for cells on laminin, adhesion forces and cell contractility triggered by integrin-mediated adhesion signaling strongly affect the outcome of epithelial cell spheroid formation . The impact on the cell area is more pronounced than in siControl cells . After e above, ,25. Blebol cells A–C.Pkhd1 cells, inhibition of cell contractility on day 1 or 2 significantly increased the proportion of correctly polarized spheroids (group 2) and strongly counteracted formation of cell aggregates (group 5) (Pkhd1 cells as compared to siControl-treated cells. With respect to cell aggregates (group 5), significant differences were lost, while there was no full recovery of correctly polarized, complete spheroids (group 1). This may be a consequence of the fact that blebbistatin by reducing actomyosin contractility also inhibits cell division. Thus, in the early stages of spheroid formation, transient reduction of cell contractility can at least in part compensate defective  adhesion signals in Pkhd1-silenced cells and allow normal epithelial morphogenesis.Next, blebbistatin treatment was applied in the spheroid assay for 24 h each, on days 1, 2 or 3. Compared to untreated sigroup 5) D. TherebThis study was designed to verify the assumption that mutation or loss of FPC, i.e., the causative events for ARPKD, leads to impaired function of renal collecting duct epithelial cells. Altered binding to and interaction with neighboring cells and extracellular matrix are considered critical aspects of epithelial dysfunction causing damage and/or failure of organs as observed in ARPKD. Therefore, we investigated if the proposed defect in environmental sensing of epithelial cells as well as consequences on epithelial morphogenesis can be demonstrated in vitro by use of a 3D cell culture model.Pkhd1-treated cells is in the same order as that seen for loss of confinement and clearly indicates altered reaction of FPC-deficient cells to the well-defined set of environmental conditions optimized to induce formation of polarized epithelia by MDCKII cells. With 34% aggregates, siPkhd1 cells are evidently not instructed properly to generate polarized epithelia. In addition, we observe 19% inversely polarized cell clusters, which are hardly ever detected in MDCKII controls, whereas there was no change in the number of cells per cluster. Furthermore, the timing of transient reduction (or loss) of FPC function correlates with defective spheroid formation. The initial two-cell stages are critical to the entire process facilitating the fate i.e., polarity and lumen formation, of cell clusters in MDCKII [To this end, we adapted the assay developed in MDCK cells for specifically such purpose by the group of Fernando Martín-Belmonte. This assay was employed by the group to show that cell confinement and spatial control of contractility are critical for centrosome positioning and lumen initiation during epithelial morphogenesis . In partn MDCKII ,26. WhenPkhd1-silencing of cells with abnormalities in cell–cell and cell–ECM interactions, the organization of the actin cytoskeleton and in cell proliferation and apoptosis and suggest a link to dysregulated extracellular-regulated kinase and focal adhesion kinase signaling [Pkhd1-treated MDCKII cells and controls is based on vinculin staining in steady-state adhesions, which provides a reasonable estimate of adhesion forces in integrin-based cell–ECM adhesion sites based on their size and the relative adhesion area [Pkhd1 cells; however, the equilibrium between actin polymerization-based cell spreading and cell contractility/tension appears to be disturbed [2), siPkhd1 cells in contrast to controls do not even fill the restricted spreading area that is provided, although these cells are not smaller in size, and upon inhibition of myosin II-based contractility, siPkhd1 cells display an even larger spreading area than siControl-treated MDCKII cells. This finding indicates enhanced contractility in siPkhd1 cells and no general problem in actin polymerization. There is no detectable difference in relative adhesion areas between siPkhd1 and siControl-treated cells, and we also did not pick up alterations in size distribution of individual adhesion sites between both groups. Activation of integrins is a central factor in determining epithelial polarity [Pkhd1 cells which are directly linked to an altered force balance. The positioning of centrosomes, as determined on crossbows (1-cell stage) and by the relative nuclear to centrosomal distance (two-cell stage), is less precise, and most importantly, limitation of actomyosin contraction in the early stage of spheroid formation is critical to the rescue of epithelial morphogenesis.An effect of FPC on cell adhesion has been considered previously. In particular, the group of Patricia D. Wilson analyzed aspects of adhesion signaling in conditionally immortalized epithelial cells from normal human fetal collecting tubules and ARPKD cyst-lining epithelia. Their analyses, which are difficult to interpret based on the data provided, suggest altered timing and intensity of focal adhesion kinase and c-Src signaling upon attachment of cells to ECM and enhanced spreading of FPC-deficient cells on collagen . Mai andignaling . Puder aignaling . Our appion area ,21. In cisturbed ,30,31. Ipolarity , and corpolarity . Analyzipolarity ,24. Furtpolarity . Thus, wIn summary, correct interpretation of environmental cues is a key feature of epithelial cell function and critical to integrity of epithelia. Upon loss of FPC, sensing of epithelial cells is modified and contributes to progressive defects of epithelial function in ARPKD. Compounds that interfere with enhanced adhesion  may prove useful to delay progress of the disease. In combination with image analysis based on supervised learning strategies , the adhMadin-Darby canine kidney cells II  were purchased from European Collection of Authenticated Cell Cultures (ECACC) and expanded, and stocks with low passage numbers were stored on liquid nitrogen (cells used between passage 3–10). MDCKII were cultured in minimal essential medium  containing 5% fetal bovine serum , 200 mM L-glutamine  and 1% penicillin/streptomycin (Biochrom) and split every 3–4 days in ratios 1:15 to 1:20.6 cells were nucleofected with 100 pmol siRNA in nucleofection mix using the Amaxa Nucleofector 2b device (Lonza) and cultured for two days before start of experiments. For analysis of 1-cell polarization and 2-cell states, cells were treated in parallel with 300 nmol of the CETN1-GFP-plasmid to visualize centrosomes [Pkhd1) and control treatment (siControl), verified sequences were taken from Zhang et al. [Pkhd1 (sense) aagcaucaaauccgaguccgu and siControl (sense) cguacgcggaauacuucgatt with corresponding antisense RNAs, respectively, and siRNAs purchased from Ambion  as custom Silencer Select.siRNA protocol: MDCKII cells were split 1:8 two days before treatment. For transfer of siRNA, cell suspensions were treated with Amaxa Cell Line Nucleofector Kit L  according to manufacturer’s manual. In brief, 0.5 × 10trosomes . For fibg et al. , siPkhd1shRNA adapted protocol: MDCKII cells were split 1:8 two days before treatment. Cells were stably transfected with Tet repressor coding plasmid pcDNA6/TR by reverse transfection using Lipofectamine 2000  and blasticidine selection (5 µg/mL).shRNA constructs  were cloPkhd1_1 (fw) tttgaagcatcaaaatccgagtccgtctcgagacggactcggatttgatgctttttttsh     (rev) ctagaaaaaaagcatcaaatccgagtccgtctcgagacggactcggattttgatgcttPkhd1_2 (fw) tttggcttctatggaaaccctttcactcgagtgaaagggtttccatagaagctttttsh     (rev) ctagaaaaagcttctatggaaaccctttcactcgagtgaaagggtttccatagaagcPkhd1_3 (fw) tttgcgcagacacccattgtttaccttcaagagaggtaaacaatgggtgtctgtttttttsh     (rev) ctagaaaaaaacagacacccattgtttacctctcttgaaggtaaacaatgggtgtctgcgPkhd1_4 (fw) tttgcgtgccagttcaccgtttggattcaagagatccaaacggtgaactggcatttttttsh     (rev) ctagaaaaaaatgccagttcaccgtttggatctcttgaatccaaacggtgaactggcacgControl (fw) tttgaatcgaggtattccgcgtacgctcgagcgtacgcggaatacttcgatttttttsh     (rev) ctagaaaaaaatcgaagtattccgcgtacgctcgagcgtacgcggaatacctcgattPkhd1-mRNA were calculated applying the ΔΔCT method and using the geometric average of three out of four reference genes [Gapdh) [Ubb) [B2M) [Hprt) [Pkhd1 (exon 42/43-44) (fw) tgcactgctagtgggtacag, (rev) ccctcggtgccagaaatact, Gapdh (fw) aacatcatccctgcttccac, (rev) gaccacctggtcctcagtgt, Ubb (fw) tcttcgtgaaaaccctgacc, (rev) ccttcacattctcgatggtg, B2M (fw) tcctcatcctcctcgct, (rev) ttctctgctgggtgtcg, Hprt (fw) gcttgctggtgaaaaggac, (rev) ttatagtcaagggcatatcc.Verification of knockdown: Efficiency of knockdown was controlled by quantitative realtime (qRT)-PCR. Two days after nucleofection, mRNA of siRNA treated cells was isolated using RNeasy Mini Kit and RNase free DNase Set . Reverse transcription of 500 ng mRNA  was followed by qRT-PCR with the Quantifast SYBR Green PCR Kit (Qiagen) on the 7900 HT Fast Real-Time PCR System . Relative amounts of ce genes ; glycera [Gapdh) , ubiquith) [Ubb) , β2- micb) [B2M)  and hypo) [Hprt)  as detecMDCKII cells were lysed directly in 65 °C prewarmed 2x Laemmli-Buffer (150 µL/75 cm²) and analyzed using 4–15% gradient gels  and pre-stained molecular weight markers (PageRuler Prestained Protein Ladder (#26616), 10 to 180 kDa, and HiMark Pre-stained Protein Standard (LC5699), 30 to 460 kDa; Thermo Fisher Scientific). Transfer was performed in Towbin Buffer . For immunodetection, rabbit anti-fibrocystin , mouse anti-vinculin , and rabbit anti-myosin IIa  were used. Secondary antibodies, IRDye 800CW goat anti-rabbit IgG (H+L)  and IRDye 680RD goat anti-mouse IgG (H+L) (1:5000) allowed quantitative fluorescence detection via the Odyssey Fc Imaging System . Signal intensities were determined by using Image Studio Software (5.2) and FPC protein normalized using geometric means of vinculin and myosin II signals to adjust protein load.4 per well, were seeded in 4-chamber system (CYTOO) allowing four independent conditions per chip and grown in MEM containing 2% FBS, 200 mM L-glutamine and 1% penicillin/streptomycin. After 4 h, half of the medium was replaced by MEM containing, in addition, 5% matrigel . Within 3 days, single cells adhering to the micropattern divided several times and formed spheroids of 16–20 cells. Optional blebbistatin treatment  was performed for 24 h, on either day 1, 2 or 3 of spheroid growth and terminated by medium change. Spheroids were washed twice with 1 × PBS, fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.25% Triton-X-100 in PBS for 12 min, blocked for one hour with 5% normal donkey serum  in PBS, stained with antibodies and dyes (as detailed below) and mounted using Shandon Immu-Mount (Thermo Fisher Scientific). Monoclonal antibodies were mouse anti-gp58 [The spheroid assay was performed on micropatterned chips  with disc-shaped micropatterns of 700 and 1600 µm² according to manufacturer’s manual and . In brienti-gp58  (1:200) nti-gp58  (1:8000)2, treated as above and stained with mouse anti-γ-tubulin , Alexa Fluor 488 donkey anti-mouse IgG (H+L)  as well as phalloidin and DAPI (see above). For visualization of micropattern, ECM coating was labeled using a combined of collagen I (see above) and fibrinogen-Atto425 . 2-cell states: Forty hours after nucleofection with siRNA, cells were synchronized for ten hours on G2/M-checkpoint using CDK1-Inhibitor RO-3306 . After eight hours of RO-3306 treatment, cells were seeded on micropatterned chips analogous to spheroid experiments and allowed to adhere in the presence of RO-3306 for another two hours. Thereafter, RO-3306 was removed, and a further six hours later, the bulk of cells had completed cell division. Cells were fixed as described and stained as described for 1-cell polarization.1-cell adhesion: Cells for single-cell adhesion were seeded on micropatterned chips analogous to the spheroid assay. After four hours, cells were fixed as above and stained for adhesion analysis with mouse anti-vinculin , Alexa Fluor 555 goat anti-mouse IgG (H+L)  as well as phalloidin and DAPI (see above). 1-cell polarization: Cells were seeded onto crossbow-shaped micropatterns of 1100 µmImages were acquired by using the AxioObserver Z1 microscope with the 63× Plan-Apochromat (NA 1.4) oil objective, the AxioCam MRm Rev.3 camera and the software package AxioVision version 4.8.2. . Filter sets used for related staining were (i) filter set 38 HE: gp58/γ-tubulin, Alexa Fluor 488; (ii) filter set 43 HE: gp135, Atto 550 and vinculin, Alexa Fluor 555; (iii) filter set 50: phalloidin, Alexa Fluor 660, (iv) filter set 49: DAPI (filter sets 1–4 from Zeiss) and (v) CFP ET Filterset F46-001 using the “CircStat” Circular Statistics Toolbox [To compare parameters from three independent experiments, 2-way ANOVA with Tukey’s multiple comparison was employed, with the exception of treatment in 2-cell states, where a non-parametric  Toolbox , whereinThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. ImageJ macros used for image analysis are provided under GNU GPLv3 license on request.