Saccharomyces cerevisiae. We started from foreign  core promoters as templates. They are, usually, unfunctional in yeast but can be activated by extending them with a short sequence, from the CYC1 promoter, containing various transcription start sites (TSSs). Transcription was modulated by mutating the TATA box composition and varying its distance from the TSS. We found that gene expression is maximized when the TATA box has the form TATAAAA or TATATAA and lies between 30 and 70 nucleotides upstream of the TSS. Core promoters were turned into stronger promoters via the addition of a short UAS. In particular, the 40 nt bipartite UAS from the GPD promoter can enhance protein synthesis considerably when placed 150 nt upstream of the TATA box. Overall, we extended the pool of S. cerevisiae promoters with 59 new samples, the strongest overcoming the native TEF2 promoter.Promoters are fundamental components of synthetic gene circuits. They are DNA segments where transcription initiation takes place. New constitutive and regulated promoters are constantly engineered in order to meet the requirements for protein and RNA expression into different genetic networks. In this work, we constructed and optimized new synthetic constitutive promoters for the yeast  Saccharomyces cerevisiae, the baker’s yeast, was the first eukaryote whose genome was completely sequenced [S. cerevisiae has been used in brewing, winemaking, and baking [S. cerevisiae has become a widely adopted chassis for genetic circuits due to its short growth cycle, simplicity of large-scale cultivation, and, above all, the easiness of genomic manipulation [S. cerevisiae cells into factories for the production of valuable chemicals such as drug precursors [equenced . Traditid baking . More repulation . These pecursors  and natuecursors .S. cerevisiae RNA polymerase II-dependent promoters are characterized by four main elements: The upstream activating sequence (UAS), the TATA box, the transcription start site (TSS), and the 5′UTR (untranslated region). Each of the first three elements can be present in multiple copies [The promoter is the place on the DNA where transcription starts. Hence, it represents a fundamental component of synthetic gene circuits. A set of e copies . The core copies .S. cerevisiae, such as the enhancers in higher eukaryotes, are bound by activator proteins that recruit the promoter RNA polymerase II together with the general transcription factor [The UASs in n factor . UASs arn factor .A consensus sequence for the yeast TATA box has been proposed by Basehoar et al.  as TATAWS. cerevisiae play an important role into synthetic gene circuits since they guarantee a relatively stable protein (or RNA) expression level under different conditions [S. cerevisiae cells and could also undergo homologous recombination with their original copy in the yeast genome.Constitutive endogenous promoters in nditions . Howevernditions  or buildnditions ,20. Thesp”) has been reported to be working in S. cerevisiae, as well [S. cerevisiae for transcription activation [To achieve orthogonality, either fully synthetic or foreign promoters shall be adopted. Viral promoters, for instance, are strongly active in mammalian cells and the cytomegalovirus promoter  and studied how to modulate its strength by: (1) Using different TATA boxes obtained by mutating, in each position, the strong TATAAAA sequence; (2) varying the distance between a selected, moderately strong, TATA box (TATATAT) and the TSS; (3) removing nucleosomes from the promoter sequence; (4) placing UASs (of variable length) upstream of the core promoter; (5) varying the distance between the UASs and the TATA box. The best results from each of these analyses were used together to improve the transcription initiation rate of both the strongest and the weakest core promoter in our initial library.S. cerevisiae genome, we found out important rules for designing and placing the sequences of both TATA boxes and UASs in order to modulate gene expression in yeast. Thus, our promoters and, more in general, our results can be used to fine-tune the working of synthetic transcriptional networks and metabolic pathways in S. cerevisiae.In addition to building, overall, 59 new yeast synthetic promoters that shared a minimal homologous region with the S. cerevisiae, by two viral promoters commonly used in mammalian cells—pCMV [S. pombe NMT1 promoter (no message in thiamine) [S. cerevisiae  and, therefore, represent a potential genomic burden that might have a negative effect on the promoter transcription efficiency. Both versions of pSV40 were unfunctional, whereas pCMVfull and the reduced CMV and NMT1 promoters turned out to be very weak, by expressing only 4.7, 1.5, and 2.3% of the fluorescence driven by the strong GPD promoter  . As for oter see .S. cerevisiae. Core promoters retain only the elements strictly necessary for RNA polymerase II binding without the help of any activator proteins. Therefore, we reasoned that the main hurdle to gene expression by foreign core promoters was the short distance between the TATA box and the TSS. At least 40 nt between a TATA box and a TSS are considered to be necessary to allow transcription initiation. It should be noted, though, that Redden et al. [As mentioned in the Introduction, foreign promoters are usually not activated in n et al.  showed tn et al. , 21 nt  minimal core promoters starting with a TATA box. Hence, with the only exception of pCaMV35Score (see below), from the promoters published in [CYC1 promoter, where six TSSs have been detected. In a previous work from our lab [S. cerevisiae promoters can be successfully built by placing pCYC1noTATA downstream of a TATA box-like sequence . In the extended version of pCMVcore, pCMVcore* (from now on we will indicate with a “*” any promoter extended with pCYC1noTATA) the TATA box is able to activate all six TSSs since the TATA box-TSS distance varies from 51 to 93 nt. In contrast, pSV40core* would fail to start transcription from the TSS at position 35 and 43 , whereas pNMT1core* activates only the TSS at position +1 (116 nt distant from the TATA box). As shown in CYC1core and ACT1 promoter  and a longer one also derived from the viral SV40 promoter (we will refer to it simply as pSV40) based on little changes on the sequences in  . Overalto 24 nt   we divided our 26 core promoters into eight groups according to their fluorescence expression . However, only the TATAAAG-containing promoter is significantly different from the other two promoters in Group 4. Thus, we decided to assign pSVcore*(TATAAAG) to a different group (number 3), that contains no other instances. The two strongest TATA boxes (the above-cited TATAAAA together with TATATAA), which forms Group 1, gave an average fluorescence level corresponding to about 91% of that of the constitutive ACT1 promoter. The only promoter in the second group, pSVcore*(TATATAC), is roughly as strong as the core CYC1 promoter [S. cerevisiae [Overall, we constructed 25 variants of the pSV40core*(TATAAAA) via single or multiple mutations on the seven nucleotides belonging to the TATA box. The highest mean fluorescence value was detected from TATAAAA, the lowest  from TATTAAT. By making a statistical analysis based on both one-way ANOVA and the two-sided Welch’s sion see A. We arepromoter  that retpromoter . From threvisiae .From our results, we can make interesting considerations about the effects of diverse mutations on different positions along the TATA box.As for single point mutations on TATAAAA, the least detrimental one was the change of an adenine to a thymine (T → A) at position 5, which did not significantly alter the core promoter strength.We checked all the possible three mutations at position 7. Each of them induced a more consistent decrease in fluorescence expression, the highest (68%) was due to the C → A substitution. Interestingly, this result is consistent with . In contWe made each of the possible three point mutations also at position 1. The decreases in fluorescence were significantly higher with respect to those concerning position 7. In particular, the G → T mutation at position 1 reduced fluorescence of even 84%. Single mutations at position 2, 3, and 4 affected fluorescence in a similar way to those at position 1 see . MutatioDouble and triple simultaneous mutations along TATAAAA confirmed the trend of reducing promoter strength with a fluorescence decrease ranging from 76 to 86% see . The onlLooking more in detail at the different kinds of point mutations, we saw that T → A is responsible for a strong decrease in fluorescence expression, unless it takes place  at position 5. In particular, a simultaneous T → A mutation at both positions 5 and 6 returned an 83% fluorescence repression see B, pointiIn general, a TATA box mainly contains adenine and thymine but a single cytosine or guanine can be found in natural TATA boxes. The insertion of a single “C” or “G” into TATAAAA caused a reduction in fluorescence, though without following a precise pattern. At position 1, C → T is more favorable than G → T (and even A → T), whereas at position 7, G → A is more tolerated than C → A. In contrast, C → A has a relatively low effect if carried out at position 7 of TATATAA, whereas G → A and T → A have an apparent negative influence on gene expression see .Overall, we have seen how simple modifications on the TATA box of a synthetic core promoter span a 9.3-fold dynamic range in gene expression. In our analysis, TATAAAA and TATATAA resulted in being strong signals, whereas the weakest was TATTAAT that, interestingly, does not contain any cytosine, guanine or thymine at position 6—a feature that seemed to attenuate considerably DNA transcription. Moreover, differently from what was present in the literature, we found out that a thymine and a guanine at the seventh position cause a strong decrease in gene synthesis. This might indicate that context effects due the nucleotides directly upstream and downstream of the TATA box play a so far not understood role in DNA transcription, which needs further study.After ranking the TATA box heptamer according to the fluorescence they drove from pSV40core*, we looked for the optimal distance between the TATA box and the TSS to maximize gene expression.A TATA box is believed to activate only TSSs that are placed from 40 up to 120 nt downstream of it . HoweverFor this new analysis, we selected the configuration of pSV40* that contained the moderately weak TATATAT box (roughly 25% as strong as TATAAAA).We constructed nine variants of pSV40*(TATATAT) by varying the distance between the TATA box and TSS from −129 to +12 nt, being −90 nt the original one . As shown in S. cerevisiae due to a non-optimal spacing between the TSS and TATA box. In contrast, promoters where TATATAT was −29, −39, −49 or −69 nt far from the TSS showed up to a ~2.5-fold increase in the fluorescence level of the original pSV40* (where TATATAT is 90 nt upstream of the TSS). The variant whose distance was −39 nt showed a lower mean fluorescence level. However, it was not significantly different in statistical terms from the other three. On the whole, our analysis shows that the region where the TATA box should lie in order to achieve maximal transcription goes roughly from position −30 to −70 with respect to the TSS.Too short or too long distances  determined low fluorescence expression, which confirmed the initial conjecture about the inoperativeness of foreign promoters in As a further attempt to improve the strength of our new synthetic promoters, we tried to remove possible nucleosomes from their sequences ,39. In tS. cerevisiae, UASs should lie at a proper distance from the TATA box, i.e., between 100 and 1400 nt [After finalizing the construction and the investigation of our core promoters, we built on them novel hybrid promoters by placing an UAS upstream of their TATA box. UASs recruit activators of different strengths and, therefore, can increase considerably the transcription initiation rate of a promoter. Moreover, in  1400 nt . For thi 1400 nt .GPD(long) (532 nt) [TEF1(long) (203 nt) [GPD(40nt) [TEF1(RAP1bs) [TEF2(59nt) [FEC built by Redden et al. [Initially, we explored the effect of different UASs on DNA transcription from pSV40*(TATATAT−49) again. We chose the long UAS-plus-spacer sequence UAS(532 nt)  and UASTF1(long) 3 nt [20,(203 nt) . In addiPD(40nt) ; the 20 (RAP1bs) ; and theF2(59nt) . Finallyn et al. . All theTEF2(59nt) (1.65-fold increase), the best one UASGPD(long) (2.45-fold increase). However, in statistical terms, UASGPD(long), UASGPD(40nt), UASTEF1(long), UASTEF1(RAP1bs), and UASFEC did not show any significant difference, pointing out that the length of synthetic promoters can be shortened remarkably by considering only endogenous activator binding sites as UASs (see Each UAS contributed to an increase in the fluorescence expressed by pSV40*(TATATAT-49). The least performant one was UASUASs see .GPD(40nt) 150 nt upstream of the pSV40*-45t, which resulted in a 1.91-fold increase in the promoter strength  to the TATA box.FEC and a core promoter was enough to achieve a very high fluorescence level. This short distance permitted, as well, obtaining minimal synthetic promoters. We placed, therefore, UASGPD(40nt) just 30 nt upstream of the TATA box of pSV40*(TATATAT-49). Compared to the bare pSV40*(TATATAT-49), the increase in fluorescence was modest (1.36-fold), whereas a 150 nt spacer guaranteed a 2.33-fold enhancement. Therefore, a too-short distance between UAS and TATA box cannot be regarded as a universally valid strategy to enhance the promoter strength strongly  150 nt upstream of the TATA box.In the previous analyses, we found that the activity of SV40*-based promoters can be improved using the heptamer TATAAAA (or TATATAA) as a TATA box, setting the distance between the TATA box and the TSS to 49 nt, and placing the short UASGPD(40nt) and the TATA box to 30 nt could give a considerable enhancement in gene expression.According to our initial measurements, MLPcore* and pTKcore* were the strongest and the weakest synthetic core promoters obtained by extending their native sequences with pCYC1noTATA. In order to see if our findings had general validity, we modified both MLPcore* and pTKcore*, where necessary, with the same changes that were effective on pSV40*. Moreover, we checked if a reduction, on both promoters, of the distance between UASGPD(40nt) either 30 or 150 nt upstream of the TATA box. In the former case, no significant difference was detected with respect to the original MLPcore*, whereas the latter provided a 1.77-fold increase in fluorescence expression. Moreover, the fluorescence intensity expressed by UASGPD(40nt)−150nt-MLPcore* exceeded that of the strong yeast constitutive TEF2 promoter . Thus, we constructed two variants of this promoter simply by adding UASoter see  such thaGPD(40nt) from the TATA box induced a huge improvement in the promoter performance: 17.54- and even 27.45-fold, respectively  is very different from the optimal one and had to be replaced. In this case, both configurations with 30 and 150 nt distancing UASvely see B. TherefS. cerevisiae synthetic promoters based on foreign template sequences coming from viral and eukaryotic cells.In this work, we constructed a large library of overall 59 new Foreign promoters, in yeast, are either unfunctional (such as pSV40) or poorly performant  due to the absence of the activators necessary to recruit RNA polymerase II and the short distance between the TATA box and the TSS.S. cerevisiae by extending them with an 87 nt long sequence that included the whole yeast CYC1 promoter 5′UTR, where at least six TSSs are present. This guaranteed a sufficient distance between the TATA box of the foreign promoter and some TSSs of CYC1 promoter to initiate transcription. We managed to make foreign promoters work in S. cerevisiae [Our experiments permitted us to confirm—as already pointed out in —that therevisiae . Moreoverevisiae . By carrGPD, TEF1, and TEF2 promoters can highly enhance promoter activity, especially when placed 150 nt upstream of the TATA box. In contrast, a much lower distance (30 nt) does not appear to be always effective.These initial experiments allowed the construction of 47 core promoters. We turned some of them into stronger synthetic promoters with the addition of an UAS. Importantly, we showed how short natural UASs from the constitutive TEF2 promoter. Additionally, the usage of a foreign template lowers the risk of homologous recombination though both UAS and 5′UTR are taken from endogenous promoters and, therefore, prevent full orthogonality.Overall, most of the core promoters in our library are relatively weak with three of them showing a moderate strength. The addition of a (short) UAS allowed the construction of stronger promoters, one of them overcoming the strong yeast constitutive Developing synthetic leaders and UASs represent the next step to realize libraries of synthetic orthogonal promoters spanning a wider dynamic range in gene expression that will be beneficial for many diverse applications of Synthetic Biology.The backbone for all plasmids constructed in this work was the yeast integrative shuttle-vector pRSII406  . The plaCYC1 terminator (CYC1t) [Every transcription unit realized in this work is composed of a synthetic promoter followed by the HIS tag, the yeast enhanced green fluorescent protein (yEGFP) , and the (CYC1t) . All theEach plasmid containing a novel transcription unit was assembled via the Gibson assembly method . To thisE. coli competent cells  were transformed with all the DNA fragments mixed in equimolar amount (30 s heat-shock at 42 °C) and grown overnight at 37 °C in LB (Luria-Bertani) plates  supplied with ampicillin. Plasmid extraction from bacterial cells was carried out using standard methods [ methods . All plaS. cerevisiae strain CEN.PK2-1C  kindly provided by Yuan Yingjin . Plasmids (5 μg) were linearized with the restriction enzyme StuI (NEB-R0187V) that cuts inside the URA3 marker. Genomic integration was carried out according to the lithium-acetate protocol, as described in [Each of our new plasmids was integrated into the genome of the yeast ribed in . TransfoTM Flow Cytometer . The FACS machine set-up was checked at the beginning and end of each experiment using fluorescent beads (BD FACSuiteTM CS&T Research Beads—650621). Measurements were considered as reliable only when the relative difference between the initial and final value of the peaks of the beads was not higher than 5%. Each strain was measured, at least, in three independent experiments . During each experiment, 30,000 events were recorded.Yeast cells were initially grown in a synthetic complete medium (SDC) at 30 °C, 240 RPM, for 18 h. Then, cells were 1:40 diluted. Green fluorescence was measured with a BD FACSVerset-test. In contrast, one-way ANOVA was adopted to analyze groups of more than two strains .Data from the flow cytometer was analyzed with the flowcore R-Bioconductor package . The mea


The deterioration of the physical–mechanical properties and loss of the chemical safety of plastics after consumption are topics of concern for food packaging applications. Incorporating nanoclays is an alternative to improve the performance of recycled plastics. However, properties and overall migration from polymer/clay nanocomposites to food require to be evaluated case-by-case. This work aimed to investigate the effect of organic modifier types of clays on the structural, thermal and mechanical properties and the overall migration of nanocomposites based on 50/50 virgin and recycled post-consumer polypropylene blend (VPP/RPP) and organoclays for food packaging applications. The clay with the most hydrophobic organic modifier caused higher thermal stability of the nanocomposites and greater intercalation of polypropylene between clay mineral layers but increased the overall migration to a fatty food simulant. This migration value was higher from the 50/50 VPP/RPP film than from VPP. Nonetheless, clays reduced the migration and even more when the clay had greater hydrophilicity because of lower interactions between the nanocomposite and the fatty simulant. Conversely, nanocomposites and VPP/RPP control films exhibited low migration values in the acid and non-acid food simulants. Regarding tensile parameters, elongation at break values of PP film significantly increased with RPP addition, but the incorporation of organoclays reduced its ductility to values closer to the VPP. Polypropylene (PP) is a thermoplastic polymer commonly used to manufacture kitchen utensils, automobile parts, textiles, pipes and packaging. PP was the most demanded plastic by European converters in 2018, representing 19% of the total produced resins (51.2 million tons) .The use of nanoparticles leads to a high polymer/nanofiller interphase area allowing an adequate energy transfer through the plastic matrix that undergoes stress. Nanoclays are specially used due to their low cost and commercial availability. They are laminar aluminosilicates that can enhance the stiffness and strength of the plastics through the intercalation of the polymer chains between clay galleries . Most ofThe bentonite clay is classified under the European Union Regulation 10/2011 (N° FCM 393) as a substance without restriction for food packaging. However, clay minerals need to be organically modified in order to increase their compatibility with hydrophobic polymers as PP. In this regard, a report about the conditions to use ethylene-based polymer/organically modified clay nanocomposites as food contact material from EFSA (European Food Safety Authority) has ruled that a case-by-case safety study is required for each nanocomposite as a function of the type of clay and organic modifier . TherefoRecently, release studies mainly focused on the migration of Si and Al from polymer/clay nanocomposites based on virgin LDPE, PET and PP have been reported ,18,19,20−1). Recycled post-consumer polypropylene pellets (RPPs) were provided by Comberplast, S.A . Two organically modified mineral clays (OCs) with different surfactants were used: (i) Non-polar-modified organoclay, OCN, (bulk density: 200–500 kg m−3) is a montmorillonite clay modified with a quaternary ammonium salt of tert-alkyl amine; (ii) OCP (bulk density: 300 kg m−3) is a montmorillonite with an organic modifier containing polar groups .Homopolymer grade virgin polypropylene pellets (VPP) was supplied by Petroquim, S.A  (density: 0.0905 ± 0.005 g mLClay/polymer nanocomposites films based on 50/50 VPP/RPP blend (NC) were obtained because concentrations above 50 wt % of RPP caused simultaneous and significant deterioration of all mechanical parameters see . Two typFilms were obtained by cast-extrusion in a twin-screw extruder Labtech Scientific LTE-20–40 , with a temperature profile of 180–190 °C, a screw speed of 20 rpm and 40% torque. The VPP and RPP pellets were previously dried at 100 °C, and the clays were dried at 40 °C in a vacuum oven, both for 48 h. Blends were homogenized before adding to the extruder hopper. Additionally, VPP, RPP and 50/50 VPP/RPP control films were prepared under the same conditions.−1, in a range 2Ɵ = 2–9°. The interlaminar distance (d001) was determined using Bragg’s Law following Equation (1):The clays and nanocomposites were characterized by X-ray diffraction (XRD) in order to determine the interlaminar distance in the clays and the type of structure of the nanocomposites. The equipment used was a Siemens D5000 brand X-ray diffractometer . The samples were explored in reflection mode using an incident Cu-Kα radiation and a step of 0.02° min−1 under nitrogen atmosphere. The decomposition initiation temperature at 5% mass loss (Tonset), the temperature at the maximum degradation rate (Td) and the mass loss at Td were determined.The thermal stability of the samples (6–8 mg) was determined in a Mettler Toledo Gas Controller GC20 Stare System TGA/DSC equipment using a heating program from 30 to 600 °C with a heating rate of 10 °C min−1 under nitrogen atmosphere. The melting (Tm) and crystallization temperatures (Tc), melting (ΔHm) and crystallization enthalpies (ΔHc) and the percent crystalline fraction were registered. The crystallinity was calculated from Equation (2), relating the PP melting heat (ΔHm) of the sample and the theoretical melting heat of 100% crystalline polypropylene (ΔH100):c is the percentage of crystalline fraction (%), ΔHm is the melting enthalpy of the sample (J g−1), ΔH100 is the melting enthalpy of fully crystalline polypropylene equal to 206.7 J g−1 [PP is the mass fraction of polypropylene in the film (%). The results of thermal parameters and crystallinity were reported as the mean and standard deviation of two measurements.DSC analysis was carried out in a calorimeter Mettler-Toledo model STAR 822e  coupled to a cooling unit . The samples in aluminum capsules (6–8 mg) were subjected to two thermal scans, heating from 25 to 250 °C and cooling from 250 to 25 °C, at a constant speed of 10 °C min−1. The results were reported as the mean and standard deviation of 15 measurements.Tensile parameters of the films such as Young’s modulus (YM), tensile strength (TS) and elongation at break (EB) were determined in a Zwick Roell universal machine  according to the ASTM D882 normative. Specimens with dimensions 16.5 cm × 2.4 cm were cut and conditioned at 23 °C for 48 h in a desiccator with RH of 50% ± 10%. The separation distance between the jaws was 50 mm and the crosshead speed was 500 mm min2 L−1. Used as acidic, non-acid hydrophilic and fatty food simulants were 3% acetic acid, 10% ethanol solutions and isooctane, respectively. Tests were conducted at 40 °C for 10 days in the acid and non-acid simulant, and at 20 °C for 48 h in the substitute fatty food simulant according to the substitute conventional conditions for isooctane of UNE EN 1186 normative.Overall migration tests were conducted according to EU Regulation N° 10/2011 and UNE-EN 1186 normative. The nanocomposites that reached the best tensile performance, VPP, RPP and VPP/RPP 50/50 control films, were analyzed. Double-sided, total immersion migration tests were conducted using an area-to-volume of food simulant ratio of 6 dm2 was quantified by gravimetry. The results were reported as the mean and standard deviation of four measurements.After the contact time, each food simulant was transferred to dry crucibles that were previously weighed. Food simulants were evaporated until reaching constant mass and the overall mass that migrated per dmp-value less than 0.05 indicated significant differences in the measurements between samples and were highlighted with different superscript letters.Data were analyzed through an analysis of variance (ANOVA) and Fisher’s multiple range test using the Statgraphic plus 5.1 program. The experimental design was the random type where a Nanocomposites presented a combination of intercalated and tactoid structures except nanocomposites containing OCN clay at 1 and 3 wt %, whose XRD diffractograms evidenced only intercalated structures indicating better nanoclay dispersion. Despite differences in polarity between PP and clays, intercalated structures were obtained by high shear and dispersive forces during extrusion. The highest hydrophobicity of the OCN organic modifier promoted greater compatibility with PP chains and their better intercalation between clay mineral layers. As is shown in The reduction of the interlaminar distance can be attributed to partial decomposition of the organic modifier confined between clay mineral layers, as it has been reported for clay/polymer nanocomposites ,23. ThisOn the other hand, the diffractogram of pure OCP presented the characteristic peak of this clay at 3.68° . MeanwhiDuring the first heating process, VPP film presented a unique endothermic transition at 164.5 °C, corresponding to the melting point of this polymer . RPP-basBesides, the melting peak of PP can be associated with homopolymer or copolymer polypropylene in the post-consumer material, considering both polymer grades have similar melting behavior . Regardim1) required for its melting  of RPP film indicated a higher PE proportion in this sample, as was expected. During the cooling process, VPP film showed a single exothermic transition at 113.6 °C corresponding to the crystallization from the molten polymer . MeanwhiThe slight differences between enthalpies of the thermal transitions in the nanocomposites can be associated with heterogeneities of the recycled polymer batch in regards to pollutant composition and molar mass distribution.TGA and DTG curves are shown in onset values, with a less significant effect on nanocomposites with OCP. These results agreed with (i) the lower Tonset for pure OCP (267.2 °C) compared to OCN (296.4 °C), indicating that earlier degradation of the organic modifier of OCP accelerated the decomposition of the nanocomposite, and (ii) the organic surfactant of pure OCP degraded with a maximum rate at a lower temperature (299 °C) than OCN (327 °C)  . Both oronset values when clay concentration was increased. Meanwhile, Td remained practically unchangeable in the nanocomposites compared to the control VPP/RPP blend when 1 wt % of the nanoclay was incorporated but decreased at higher concentrations (3 and 5 wt %). This fact can be attributed to a higher amount of the nanofiller poorly attached to the PP matrix at a higher concentration of clay, which tended to decompose, although less significantly for OCN with a more hydrophobic organic modifier. Nonetheless, regardless of the type of clay, all nanocomposites had mass losses lower than the VPP/RPP control film at the maximum degradation rate.In addition, regardless of the type of clay, the decomposition tended to start at lower TThe tensile parameters of the 50VPP/50RPP blend were between values of the VPP and RPP films. Increasing RPP concentration deteriorated the mechanical performance of the films, as was expected . The YM Deformation at break values drastically increased as a function of RPP concentration in films without clay, showing a 2.8 and 9.6-fold increase with respect to virgin material for VPP/RPP 50/50 blend and 100% RPP sample, respectively. This fact can be associated with the presence of ductile polymers such as polyethylene in the RPP. For example, EB values above 800% have been reported for several LLDPE and HDPE grades ,29,30.On the other hand, mechanical parameters of the nanocomposites depended more on the resulting polymer/clay structures formed than their crystallinities, which were similar at all concentrations see . YM tendDeformation at break values of the nanocomposites were significantly lower than EB values of VPP/RPP blend and closer to the VPP deformation . These rBased on these results, the NC-1OCN nanocomposite had the most improved mechanical parameters, mainly elastic modulus and tensile strength, in accordance with the higher intercalation of PP chains between clay mineral layers, as it was previously observed through XRD due to the greater affinity of the hydrophobic organic modifier of OCN with PP.If recycled polymers are intended to be applied as food contact packaging materials, analyzing the effect of the recycling processes on their safety is necessary. Thus, overall migration studies of RPP nanocomposites under three different food simulants were carried out to understand their dependence on the type of packaged food. The overall migration values from VPP, RPP, 50VPP/50RPP and NC films at 1 wt % of clay, which had the best mechanical performance are shown in The overall migration from 100% VPP and RPP control films was determined in a fatty food simulant, considering that this medium caused the maximum migration due to the greater affinity between fatty simulant and PP. A higher RPP percentage in the films without clay significantly increased migration, with a 2.9 and 4-fold increase for 50% and 100% RPP samples, respectively.−2 from plastics to be used in direct contact with food, equivalent to 60 mg per kg of food occupying a volume of a cubic package with 6 dm2 of total area. Thus, RPP-based films in the fatty simulant at the conditions studied exceeded this limit. However, the EU normative (N° 10/2011 with modification 2016/1416) considers applying fat reduction factors to migration values for food categories using a D2 fat simulant at the predictable conditions of use.The current European Union Regulation (No. 10/2011 of the Commission of 14 January 2011) establishes an overall migration limit of 10 mg dmIn the fatty simulant, the incorporation of the nanoclays in the PP matrix decreased the overall migration probably due to tortuous pathways created by clay mineral layers that increased the barrier properties, and therefore, hindered migration from the polymer. This phenomenon has been similarly observed for RPET/clay nanocomposites . The nanocomposite containing OCP exhibited a more significant decrease of the overall migration in the fatty simulant, probably due to some polarity of the OCP organic modifier containing hydroxyl groups, which inhibited the interaction of the hydrophobic simulant with the nanocomposite.−2) than migration values obtained in fatty simulants. Besides, no statistically significant differences were observed between the 50VPP/50RPP control film and nanocomposites. However, it can be noted that nanocomposites with OCP showed a slight tendency to increase migration compared to films with OCN. These results highlighted the effect of the higher hydrophilicity of the OCP, which increased the affinity between the nanocomposite and aqueous simulants, causing a greater penetration of the simulant in the polymeric matrix and a higher release of their components.Conversely, overall migrations from 50VPP/50RPP control and nanocomposites films in the acidic and non-acidic aqueous simulants were far below (<1 mg dmThe nanocomposite at 1 wt % of OCN had the best thermal and mechanical performance at the studied conditions. The highest intercalation grade of PP between clay mineral layers was reached with OCN because its organic modifier is more hydrophobic. Pollutant polymer traces in the RPP drastically increased its ductility. Interestingly, nanoclay incorporation caused a reduction of the elongation at break of the RPP/VPP film, although it did not significantly increase Young’s modulus and tensile strength, which were instead reduced at high clay percentage. The latter can be due to a greater amount of tactoids or clay particles poorly attached to the polymer matrix, considering crystallinities were similar for all nanocomposites.−2, far below the limit ruled by current European legislation. Meanwhile, the overall migration of free-clay RPP-based films and the nanocomposites in fatty simulant exceeded that limit, and, although the incorporation of nanoclays exerted a barrier effect, migration values were still higher than the allowed limit. Nonetheless, fat reduction factors can be applied in an adequate fatty simulant according to current European legislation for plastics to be used in specific food categories and predictable use conditions. Finally, it is highlighted that the clay with the most hydrophobic organic modifier improved the thermal properties, the intercalation grade and some mechanical parameters of the VPP/RPP blend, but increased the affinity of the fatty simulant towards nanocomposites and overall migration to that food simulant.Conversely, the overall migration values of nanocomposites to 3% acetic acid and 10% ethanol food simulants were lower than 1 mg dm


Acute kidney injury (AKI) and chronic kidney disease (CKD) are common events after radical nephrectomy (RN). In this study we aimed to predict AKI and CKD after RN relying on specific histological aspects. We collected data from a cohort of 144 patients who underwent radical nephrectomy. A histopathological review of the healthy part of the removed kidney was performed using an established chronicity score (CS). Logistic regression analyses were performed to predict AKI after RN, while linear regression analysis was adopted for estimated glomerular filtration rate (eGFR) variation at 1 year. The outcomes of the study were to determine variables correlated with AKI onset, and with eGFR decay at 1 year. The proportion of AKI was 64%. Logistic analyses showed that baseline eGFR independently predicted AKI . Moreover, AKI , baseline eGFR , and the presence of arterial narrowing  were independently associated with eGFR decline. Our findings showed that AKI onset and eGFR decline were more likely to occur with higher baseline eGFR and lower CS, highlighting that RN in normal renal function patients represents a more traumatic event than its CKD counterpart. Acute kidney injury (AKI) and chronic kidney disease (CKD) are two possible sequelae following radical nephrectomy (RN) . Post-suAlthough numerous efforts have been made by clinicians to better understand and predict the development of AKI and CKD after RN, several problems remain unsolved. One of the most intriguing arguments is that AKI and CKD occurring after surgery should be considered differently in respect to other clinical settings when renal function decline is sharply compromised but not related to an acute loss of nephron mass . At the Thus, to address these points, we carried out an in-depth analysis on the clinical and pathological characteristics of 144 consecutive RN cases in predicting AKI and CKD.2 (G1), 60 mL/min/1.73 m2 (G2), and 45 mL/min/1.73 m2 (G3a and G3b-4). The study consists in two different but related parts. In the first one, the primary outcome is to identify clinical variables correlated with the AKI onset after radical nephrectomy; in the second one, the primary outcome is to determine the eGFR decay after one year from surgery. In both cases subsequent analysis was carried out to model the eGFR decay  with respect to the clinical variables.We performed a retrospective cohort study of patients who underwent RN due to the presence of a kidney mass suspected of malignancy. The following data were considered: age, gender, body mass index (BMI), TNM staging, Fuhrman grading, hypertension, diabetes, and medical therapy (ACE inhibitors (ACEi), angiotensin II receptor blockers (ARBs), calcium antagonists, beta blockers, and diuretics). Serum creatinine (s-Cr) values (Kinetic Picrate standardized COBAS C 800) for IDMS) were collected before surgery (t0), at 24 and 48 h after surgery, and at dismissal , as well as after one year (ty) to detect renal function fluctuations and the subsequent risk of AKI, which usually presents within 48 h of surgery ,10. Base00 for IDThe cohort consisted of 144 consecutive patients diagnosed with a kidney mass who were candidates for elective radical nephrectomy (2009–2019). Exclusion criteria were as follows: (a) age < 18 years, (b) CKD category G5 according to KDIGO guidelines , (c) solPostoperative AKI was diagnosed according to s-Cr increase 72 h after surgery based on the KDIGO criteria . AccordiHistological evaluation was performed on two dedicated specimens of kidney tissue derived from the unaffected part of the removed kidney (at least more than 3 cm away from the tumor). All histological samples were examined by the attending pathologist in order to identify signs of medical nephropathy. A slide with a specimen of approximately 15 × 20 mm containing both the medulla and cortex from normal tissue was prepared from histology. Then, two parallel lines were drawn on the normal kidney tissue with a marking pen to simulate biopsies using a 16-gauge biopsy core. A Nikon Eclipse 80i microscope  with Pan Fluor was used at different power to evaluate an area of about 20 × 1.3 mm that was randomly chosen from one side of the line.All samples were buffered, formalin fixed, paraffin embedded, and 4 mm thick kidney sections were stained with hematoxylin and eosin. We then analyzed samples for each subject using the chronic lesions score described by Remuzzi et al. in 1999 . This scAnalyses were carried out to detect potential correlations between the outcomes of interest  and other independent variables. A linear regression model was used to predict continuous variables , while we adopted the non-parametric Wilcoxon test (two groups) and Kruskal–Wallis test (more than two groups) for comparisons of continuous variables between groups. Wilcoxon test with Holm’s correction was used for post hoc test comparisons and Fisher’s exact test was performed for comparisons between categorical variables. Binomial logistic regression was used to identify possible predictors of AKI onset. The significance level for all the analyses was set at 0.05. All analyses were performed using programming language for statistical computing R version 3.6.3  , R packaDescriptive characteristics of the patient population are reported in p = 0.7) or Fuhrman grade (p = 0.3). The median hospital stay after the surgery was 3 days (3–3). After surgery, 69% of patients demonstrated a rise in serum creatinine, which classified them as affected by AKI, according to KDIGO criteria. It is important to underline that the majority of these patients were classified as stage 1 (58%), with only a small percentage of stage 2 (8%) and 3 (3%). For the purposes of our analysis, we considered AKI onset as the outcome and investigated for predictors among clinical data and comorbidities. We did not observe any correlation with age, gender, intraoperative blood loss, presence of hypertension (considering the use of specific drugs), diabetes, BMI, TNM staging, Fuhrman grading, and histopathology. Logistic regression predicting AKI onset showed that baseline eGFR was a significant predictor for the CKD-EPI formula, as shown in Overall, 21% of patients had CS = 0, 35% had CS = 1, 26% had CS = 2, and 18% had CS ≥ 3. No correspondence was observed with the TNM score  1.04 for CKD-EPI). Evaluation of the eGFR trend after nephrectomy showed that patients who developed AKI had a higher pre-surgery eGFR, as shown in Linear regression analysis between the pre-surgery value of eGFR and its decay at 24 h, 48 h, and 72 h exhibited significant results . p = 0.02) and with the presence of tubular atrophy (p = 0.006) or interstitial fibrosis (p = 0.02). The results confirmed that already damaged tissue correlates with a milder eGFR decay  was found between baseline eGFR and renal function decay over 1 year. The higher the baseline value, the greater the eGFR decline. (p < 0.001). Indeed, patients who developed AKI during hospitalization showed a greater than 17% decline , and 2% of patients were classified (according to the CKD-EPI equation) as G1, 26% as G2, 58% as G3a, and 14% as G3b-4. No significant correlations regarding hypertension, diabetes, and overweight or obesity at time zero were observed. A significant correlation (decline. . AKI ons decline .p = 0.003). The post hoc test enlightened that eGFR decay for patients with CS ≥ 3 had a milder reduction of the renal function than patients with CS = 0 and 1. The four components of the CS were correlated with eGFR decline, as reported in We also observed that the eGFR decay after 1 year correlated with the CS of the removed kidney (2 displayed a median eGFR decay of 29 mL/min/1.73 m2 (20–34) and a 74% rate of AKI development. In parallel, another significant association was reported between lower CS, meaning the absence of histological damage, and a higher eGFR decline. These results are hypothesis generating. First, the absence of a strong correlation between pre-existing hypertension and AKI development could be related to good compliance with anti-hypertensive therapy by patients before surgery. Regarding obesity, a possible explanation could be that this condition likely leads to a compensation mechanism given its known effect on glomerular hyperfiltration. As far as diabetes is concerned, none of the patients had diabetic nephropathy with clear signs of kidney dysfunction and gross proteinuria or progressive kidney injury. Furthermore, the absence of correlation between AKI development and the aggressiveness of the tumor may be linked to the inability of cancer tissue to affect overall renal function, while the remaining “healthy” parenchyma tends to compensate. Conversely, the surprising association between a “healthier” kidney status before surgery and the greater risk of developing surgical AKI could be unexpected at first glance. However, as the literature has already demonstrated, this result could be explained by a compensatory mechanism already present in patients who had a previously compromised kidney status with an eGFR < 60 mL/min/1.73 m2. Hyperfiltration in the remaining kidney seems to occur progressively in the advanced stages of CKD and, thus, the acute loss of nephrons does not represent a severe traumatic event on overall renal function in comorbid patients. On the contrary, as demonstrated in the literature by the comparison between LKDs and oncological patients, surgical AKI is more likely to occur in healthy individuals with no histological damage because the total renal function is still carried out by both organs. The same observations were reported by Martini et al., who showed that partial nephrectomy induces more strain in patients with normal baseline eGFR [p < 0.001). We observed that at 12 months, 85% of AKI patients fell into a lower eGFR category with respect to their baseline, while only 15% remained within the same category. Interestingly, the median eGFR decay in patients who developed AKI was greater among those whose eGFR was ≥60 mL/min/1.73 m2. As a matter of fact, the greater the impairment of baseline renal function, the lower the median decline in eGFR over time. In addition, none of the patients with baseline eGFR < 60 mL/min/1.73 m2 developed ESRD at 1 year. Conversely, patients with normal renal function had a median decay of 28 mL/min/1.73 m2 (21–34) one year after RN. Finally, it is also important to underline that the eGFR value at discharge after RN did not correlate with the decay at one year. The chronicity score evaluated the healthy part of the removed kidney at the time of surgery and demonstrated that the absence of histological damage (CS0–CS1) was prognostically related to a greater decline at 1 year, with worsening of the eGFR category in 87% of cases. Regarding the absence of arterial narrowing, 88% of patients developed CKD, while for kidney fibrosis, this finding occurred in 81% of patients. The correlation between AKI development and eGFR decay at 12 months confirmed the hypothesis that in some cases the remnant contralateral kidney is likely unable to compensate for the removed one in the short- to mid-term, especially if eGFR was higher and no histological damage was present at baseline. These results suggest that, before surgery, both kidneys were correctly filtrating in these patients and that the remaining kidney was not able to achieve the same eGFR total value on its own, neither immediately nor at 1 year. On the contrary, when baseline eGFR was <60 mL/min/1.73 m2, most of the overall renal filtration was being performed by the contralateral kidney, and so, acute and chronic decline remained low because the contribution of the tumoral kidney was negligible. These results can be compared to those reported by Bachrach and colleagues, who quantified the contribution of the removed kidney to overall filtration ranging from 50% to less than 20% in a study involving 136 patients who underwent radical nephrectomy or nephroureterectomy and had a preoperative renal scan with calculated differential function. The compensation of the remaining kidney exceeded 12% of the expected value in the entire population and almost 15% in patients with eGFR < 60 mL/min [In the last decade the concepts of surgical post-operative AKI and CKD caused by the acute loss of nephrons have been proposed in order to distinguish them from the pathological medical conditions of AKI and CKD . In factine eGFR . Our resine eGFR . Conside0 mL/min . These d0 mL/min . We can 0 mL/min ,25. Howe2 values (for both models) indicate low accuracy and are mainly due to two factors: the small number of patients involved in the creation of the model and the biological variability that plays a key role compared to external elements [The main limitations of the study are due to its retrospective nature. The use of serum creatinine values for classifying AKI was due to the lack of other biomolecular marker levels ,26,27,28elements .Predicting AKI and eGFR decay after RN should take into consideration a combination of clinical, laboratory, and pathological features. The pathological analysis should be routinely performed in the healthy part of the removed kidney and combined with the other clinical parameters to provide tailored multidisciplinary counseling. Finally, both surgically derived AKI and CKD should be considered and classified differently with respect to their medical alter egos.


Renal cell carcinoma (RCC) is a heterogeneous group of kidney cancers. Renal capsule invasion is an essential factor for RCC staging. To develop radiomics models from CT images for the preoperative prediction of capsule invasion in RCC patients.This retrospective study included patients with RCC admitted to the Chongqing University Cancer Hospital (01/2011–05/2019). We built a radiomics model to distinguish patients grouped as capsule invasion versus non-capsule invasion, using preoperative CT scans. We evaluated effects of three imaging phases, i.e., unenhanced phases (UP), corticomedullary phases (CMP), and nephrographic phases (NP). Five different machine learning classifiers were compared. The effects of tumor and tumor margins are also compared. Five-fold cross-validation and the area under the receiver operating characteristic curve (AUC) are used to evaluate model performance.This study included 126 RCC patients, including 46 (36.5%) with capsule invasion. CMP exhibited the highest AUC (AUC = 0.81) compared to UP and NP, when using the forward neural network (FNN) classifier. The AUCs using features extracted from the tumor region were generally higher than those of the marginal regions in the CMP (0.81 vs. 0.73) and NP phase (AUC = 0.77 vs. 0.76). For UP, the best result was obtained from the marginal region (AUC = 0.80). The robustness analysis on the UP, CMP, and NP achieved the AUC of 0.76, 0.79, and 0.77, respectively.Radiomics features in renal CT imaging are associated with the renal capsule invasion in RCC patients. Further evaluation of the models is warranted. Renal cell carcinoma (RCC) is a heterogeneous group of kidney cancers comprising many histologic subtypes, with clear cell histology being the most common subtype , 2. RCC The renal capsule is a firm fibrous layer surrounding the kidney and is covered by a thick layer of perinephric adipose tissue. The fibrous layer can protect tumor from seeding or spreading to the adjacent tissue, thus a capsular invasion is an early sign of cancer spreading and reflects a tumor’s aggressiveness. Several studies –9 showedThe visual assessment of capsule invasion at imaging, however, can be challenging because RCC patients with capsule invasions may or may not invade the renal perirenal fat space –15. ReceRadiomics is a computational method to extract massive quantitative features from medical images for investigating clinical outcomes , 18. PreThis study was a retrospective study and was approved by the ethics committee of the Chongqing University Cancer Hospital (CZLS2021068-A). The requirement for informed consent was waived by the ethics committee of the Chongqing University Cancer Hospital. The study included patients admitted to the Chongqing University Cancer Hospital and diagnosed with RCC from January 2011 to May 2019. The inclusion criteria were (1) pathologically confirmed RCC after partial or radical nephrectomy surgery and (2) complete CT scan acquired within 2 weeks before surgery.A histopathological evaluation was performed with hematoxylin and eosin staining for all patients, along with immunohistochemistry when needed. The tumor histological findings were classified according to the WHO 2004 system . An assoAll data of the patients included in this study were collected from the Integrated Electronic Medical Record System of the Chongqing University Cancer Hospital, including age, sex, tumor location, maximal diameter of the tumor, Furhman stage, and lymph node metastasis.All patients underwent multi-phase enhanced CT scanning. Three different CT systems  with comparable clinical operations were used on patients upon availability. The majority of patients n = 107; 85%) were scanned using the Philips Brilliance CT 64. Table 07; 85% wFor contrast-enhanced imaging, iodixanol alcohol 80–90 mL (320 mg/mL) was injected into the elbow vein with a high-pressure syringe at a rate of 2.5–4.0 mL through a power injector. The scanning started from the top of the diaphragm to the level of the iliac wing. The CT images were acquired at three different scanning phases: (1) phase 1, unenhanced phase (UP), before the injection of the contrast agent; (2) phase 2, corticomedullary phase (CMP), 7 s after aortic enhancement exceeded 150 HU compared to baseline; and (3) phase 3, nephrographic phase (NP), 20 s after CMP.The tumors in the UP, CMP, and NP images were manually segmented by a certified associate chief radiologist with 10 years of experience, using the Philips Radiomics Tool. The largest cross-sectional region of the tumor was selected for segmentation. Then, another senior radiologist (10 years of experience) reviewed the selection to confirm or make corrections to the initial segmentation results. To evaluate the effects of the tumor margin regions, an extended area of 3 mm adjacent to the tumor boundary was automatically generated by image processing algorithms to extract the radiomics features of the peritumor region. The 3-mm margin was determined based on the clinical experience and considerations of the two expert radiologists.As seen in Fig. To evaluate the effects of the tumor regions and the tumor’s margin regions, radiomics models were built using features from the tumor region alone, the marginal region alone, and their combination. Two “combination” modes were evaluated: (1) combining radiomics features separately extracted from tumor and marginal area (marked with “T + M”); and (2) extracting features directly from the combined/integrated region of the tumor and margin (denoted by “whole region”).p < 0.05 was considered statistically significant.The least absolute shrinkage and selection operator (LASSO) algorithm  was usedp = 0.039). The number of tumors located on the left and right sides was similar. The tumor size ranged from 1.2 to 16 cm, and the tumors were smaller in the capsule invasion group . The patients in the capsule invasion group had more advanced stages (p = 0.002). According to pathology, only nine (7.1%) patients out of 126 had lymph node metastasis.This study included 126 patients with RCC; 46 had capsule invasion, and 80 had no capsule invasion. Table Figure Based on the results mentioned above, FNN was used as the primary model for the subsequent analyses with features extracted from different regions. Table p < 0.05) to the corresponding one-round result 0.74, 0.81, and 0.77, showing the stability of the FNN classifier and the radiomics model.To further evaluate the robustness of the FNN classifier, the algorithm was performed 100 times  to determine the mean and standard deviation of the ROC curves for the UP, CMP, and NP Fig. . Even whIn this study, we developed a radiomics model from CT images for the preoperative prediction of capsule invasion in RCC patients. The results show an association between radiomics features in renal CT imaging and the renal capsule invasion of RCC, and, in particular, it shows that the characteristics of tumors at the CMP stage are predictive of the capsule's invasion.The results of this study showed that the radiomics features separately extracted from the tumor or marginal regions are generally more predictive than the features extracted from the combined regions . This indicates the LASSO method may be able to select better-performing feature combinations to achieve the higher classification performance. Previous studies have studied different features among tumor, peritumoral, and necrosis areas –30. TherIn addition, as observed for the whole versus individual T and M results, the combination of the features extracted from the UP and CMP phase showed overall worse performance than using either UP or CMP alone. It suggests that these two scanning phases may have some conflicting features for FNN to effectively learn patterns. Likewise, the model performance decreases significantly when we combine the features extracted from all three scanning phases compared to using the features extracted from UP, CMP and NP alone. This may further imply the increasing redundancy of information conveyed by these mixed features, making the feature selection procedure less effective. Of note, the UP phase achieved higher performance than CMP and NP as well as their combinations. This suggests that contrast-enhanced imaging may not be necessarily beneficial for the detection of capsule invasion. Yet, previous studies have shown various results regarding the effects of enhanced/unenhanced imaging –33. ThisThis study shows no significant association between RCC capsule invasion and age, tumor localization, and lymph node metastasis. There is moderate association between the Fuhrman stage and capsule invasion, as expected, based on the definition of the Fuhrman stages , 9, 21.While previous work used simple qualitative imaging signs , 12, 15,Our study has some limitations. First, the sample size was small, and this is a single-center study. Further evaluation of the models and findings are warranted on larger datasets. Second, the radiomics models use two-dimensional images instead of three-dimensional CT scan, so its performance needs further evaluation on three-dimensional data . In addiIn conclusion, preoperative CT-based radiomics features are shown to be associated with renal capsule invasion of RCC. Renal capsule invasion may be characterized by radiomics of unenhanced CT imaging and the CMP images. Compared to traditional visual assessment of images, the radiomics model may provide a tool to aid assessment of whether a capsule is invaded, and thus to better inform clinical prognosis and patient management. Further evaluation of our findings is warranted on large datasets in future work.


Previous studies have shown that heart failure (HF) and chronic kidney disease (CKD) have common genetic mechanisms, overlapping pathophysiological pathways, and therapeutic drug—sodium-glucose cotransporter 2 (SGLT2) inhibitors.  The genetic pleiotropy metaCCA method was applied on summary statistics data from two independent meta-analyses of GWAS comprising more than 1 million people to identify shared variants and pleiotropic effects between HF and CKD. Targets of SGLT2 inhibitors were predicted by SwissTargetPrediction and DrugBank databases. To refine all genes, we performed using versatile gene-based association study 2 (VEGAS2) and transcriptome-wide association studies (TWAS) for HF and CKD, respectively. Gene enrichment and KEGG pathway analyses were used to explore the potential functional significance of the identified genes and targets.  After metaCCA analysis, 4,624 SNPs and 1,745 genes were identified to be potentially pleiotropic in the univariate and multivariate SNP-multivariate phenotype analyses, respectively. 21 common genes were detected in both metaCCA and SGLT2 inhibitors' target prediction. In addition, 169 putative pleiotropic genes were identified, which met the significance threshold both in metaCCA analysis and in the VEGAS2 or TWAS analysis for at least one disease.  We identified novel variants associated with HF and CKD using effectively incorporating information from different GWAS datasets. Our analysis may provide new insights into HF and CKD therapeutic approaches based on the pleiotropic genes, common targets, and mechanisms by integrating the metaCCA method, TWAS and VEGAS2 analyses, and target prediction of SGLT2 inhibitors. It is estimated that more than 23 million people worldwide are currently affected by heart failure (HF) , which dIt is reported that the estimated heritability ratio of CKD is about 30-50% , while HAs far as we know, the shared mechanism and risk molecular of HF and CKD have been discussed at proteomic and epigenetic levels , 19. HowThe canonical correlation analysis that uses multivariate statistics to represent genotypic and phenotypic variables based on published univariate GWAS statistics may be suitable for overcoming GWAS limitations . GenotypIn this study, we verified the results of metaCCA by combining the multiomics dataset rather than just using genome data to identify 169 disease-associated pleiotropic genes. Potential therapeutic targets of SGLT2 inhibitors shared by HF and CKD were also detected. In addition, we conduct functional enrichment and PPI network to explore the genes and therapeutic targets we identified.http://www.broadcvdi.org/. Cases refer to participants with a clinical diagnosis of HF of any etiology without inclusion criteria based on LV ejection fraction; controls were participants without HF [−1 per 1.73 m2 were defined as CKD regardless of etiology [https://ckdgen.imbi.uni-freiburg.de. We referred to every original study of these two GWAS meta-analyses to ensure all the samples in the GWAS datasets came from different populations with European ancestry. Additionally, the summary statistics have undertaken double check of genomic control separately at the individual study level and meta-analysis. Please refer to the corresponding consortium publications for more detailed information on the sample ascertainment and stringent quality control procedures.The GWAS dataset for heart failure was adopted from a meta-analysis containing 26 studies that comprise 47,309 cases and 930,014 controls of European ancestry. Its summary statistics of 8,021,999 imputed SNPs were downloaded from etiology . And itshttp://www.cog-genomics.org/static/bin/plink/glist-hg19). Only the SNPs that can be annotated were reserved for the following procedures. Second, we conducted a linkage disequilibrium (LD) to remove SNPs with large pairwise correlations based on the SNP pruning method. The SNP pruning method was proceeded by a window size of 50 SNPs and step size of 5 SNPs, which means LD was calculated between each pair of SNPs to remove SNPs in high LD; each sliding window of 5 SNPs moved forward and the process repeated until there were no pairs of SNPs with high LD [r2 > 0.2 were also removed [β and its standard error. The regression coefficient β was normalized before conducting the metaCCA analysis. Standardization was achieved according to the following equation:gp is the standard error of βgp. Both SEgp and gpβ are given by the original GWAS result; g is the number of genotypic variables, p is the number of phenotypic variables, and n is the sample number of each disease.After downloading summary statistics, we took several steps to process data for implementation of the metaCCA method. First, we combined the summary statistics to identify common SNPs between studies of HF and CKD. Then 7,754,982 overlapping SNPs of HF and CKD have completed the gene annotation for the two GWAS according to the 1000 Genomes dataset using PLINK1.9 ( high LD . SNPs wi removed . All datXY), a genotypic correlation structure among SNPs (∑∧XX), and a phenotypic correlation structure among traits (∑∧YY) [XY is constructed as the normalized regression coefficient βgp:To identify the potential pleiotropic genes, we applied the metaCCA method. The principle and algorithm of metaCCA were consistent with the description of the paper of Cichonska et al. . In shors (∑∧YY) . ∑XY is g and p are the numbers of genotypic and phenotypic variables, respectively.In the formula, ∧XX was calculated using the 1000 Genomes reference for Europeans (phase 3) as a reference representing the study population. Furthermore, the phenotypic correlation structure (∑∧YY) was computed based on ∑XY. Each entry of ∑∧YY corresponded to a Pearson correlation coefficient between the vector of β estimates from p phenotypic variables across g genetic variants. For the sample size varies between these two studies, we set the smallest sample size among the included traits as N, which is the most conservative and commonly used approach to address this issue. After calculation, the full covariance matrix (∑) can be obtained:In our study, ∑r [P value < 0.01 was used as the threshold for significance. If the P value of the canonical correlation r of any SNP was less than 2.77 × 10−8 ( = 0.01/440440), it was deemed significantly associated with HF and CKD. Similarly, genes with a canonical correlation P value smaller than 6.54 × 10−7 ( = 0.01/15302) were significantly associated with HF and CKD.The full covariance matrix was plugged into the CCA framework to produce the final genotype-phenotype association result . The corr . In thishttps://pubchem.ncbi.nlm.nih.gov/), which is an open chemistry database with 96,502,248 compositions of which 3,151,393 have been tested. We adopted SwissTarget Prediction (http://www.swisstargetprediction.ch/), a tool for target prediction according to dimensional and 3-dimensional similarity measures with known ligands, to select and to predict potential targets for four SGLT2 inhibitors by putting their chemical structures into this platform [https://www.drugbank.ca/), a unique bioinformatics and chemical informatics database containing 11,628 drugs and related chemical information, drug targets, protein data, and so on [http://www.uniprot.org/), all the SGLT2 inhibitors' related genes were normalized into consistent symbols for subsequent analysis. According to the intersection of genes identified by metaCCA and SGLT2 inhibitors' related targets, we get the Venn diagram on the website (https://bioinfogp.cnb.csic.es/tools/venny/).The chemical structures of four SGLT2 inhibitors, namely, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin, were obtained using PubChem (platform . Additiond so on . With fuhttps://vegas2.qimrberghofer.edu.au/). This method is aimed at determining the association between each SNP and each trait using original GWAS summary statistics individually [P value threshold.To make the identified genes by metaCCA more credible, we performed gene-based association analysis by using the versatile gene-based association study 2 (VEGAS2) method , which is attached to the GTEx database. Genes with corrected P values < 0.05 were identified as significant risk genes. For more information of FUSION parameters, please refer to the previous article [Transcriptome-wide association studies (TWAS) of HF and CKD were performed using FUSION software. FUSION can compute gene expression weights for different tissues by individual's genotype and gene expression data. In the current study, gene expression weights of the whole blood panel were adopted from the FUSION website  bioinformatics tool was used to evaluate these analyses. A GO term or KEGG pathway with an adjusted nificant . Visuali network . The worP < 2.77 × 10−8), and the canonical correlation r between each SNP and phenotype ranged from 0.005 to 0.013. The Manhattan plot in P value (P < 6.54 × 10−7) were identified as the potential pleiotropic genes. The canonical correlation r between genotype and phenotype ranged from 0.008 to 0.069.After gene annotation and SNP pruning, 440,440 SNPs located in 23,579 gene regions were inputted in the metaCCA analysis. Totally, 4,624 SNPs reached the Bonferroni corrected threshold (http://jvenn.toulouse.inra.fr/app/example.html). We finally identified 21 potential therapeutic targets for SGLT2 inhibitors in both heart and kidney failures, including SLC5A1, EP300, and GRIK1, which were also considered to be pleiotropic genes. The results are presented by the Venn plot in After importing four SGLT2 inhibitors' structures obtained from PubChem into the SwissTargetPrediction database, respectively, for target matching and prediction, we screened the 400 targets  for CKD and ATXN2  and FTO  for HF, indicating that metaCCA have a more statistic power. In comparison, 100 genes were determined as novel putative pleiotropic genes because these genes have never been reported to be associated with HF and CKD , positive regulation of microtubule nucleation (GO:0090063), regulation of microtubule nucleation (GO:0010968), response to heat (GO:0009408), and phagosome acidification (GO:0090383). Biological process dot plot of the top 10 GO terms of 169 putative pleiotropic genes is presented in In this study, we used two independent GWAS meta-analyses with available summary statistics of HF and CKD to perform the multivariate pleiotropic mapping. Potential therapeutic targets of SGLT2 inhibitors shared by HF and CKD were determined by the intersection of genes in metaCCA and SGLT2 inhibitors' related target analysis. Furthermore, genes determined by metaCCA have then obtained a verification using VEGAS2 analysis and TWAS analysis. We finally identified a total of 21 potential therapeutic targets and 169 putative pleiotropic genes. Moreover, we performed the functional enrichment analysis based on these results. In KEGG analysis based on putative pleiotropic genes and potential therapeutic targets, we identified several pleiotropic pathways that have been confirmed to be shared by HF and CKD, which may also play an important role in the SGLT2 inhibitors pharmacological mechanism. The result of functional enrichment provided us with a better understanding of the potential common biological pathogenesis of HF and CKD.Seven out of the twenty-one therapeutic targets have been reported to be associated with both HF and CKD. In the remaining genes, only GRIK1, FDFT1, and PFKFB3 were first reported to be therapeutic targets of HF and CKD. Previous studies have demonstrated therapeutic mechanisms for some potential pleiotropic therapeutic targets of HF and CKD identified by metaCCA and SGLT2 target prediction. For example, sodium-glucose cotransporter SGLT1  accounts for most of the dietary glucose uptake . SLC5A1 Among 169 putative pleiotropic genes, 100 genes are identified as novel, while 26 genes have been reported to be critical to HF or CKD etiology. For example, FTO is a confirmed pleiotropic gene, which encodes an N6-methyladenosine (m6A) RNA demethylase . In addiSeveral confirmed pleiotropic pathways shared by HF and CKD were also detected by enrichment analysis, including the insulin signaling pathway, the AMPK signaling pathway, and the apelin signaling pathway. These pathways may also play an important role in the SGLT2 inhibitors' pharmacological mechanism. Activation of the proximal insulin signaling pathways within cardiomyocytes increases when HF may contribute to adverse left ventricular remodeling and mitochondrial dysfunction . InsulinAdenosine 5′-monophosphate- (AMP-) activated protein kinase (AMPK) is a critical molecule in the regulation of bioenergy metabolism . It is tTwo original GWAS included cohorts with either a case-control or population-based study design regardless of the etiologies of HF or CKD. Although HF and CKD are complex clinical syndromes with various etiologies, the result of this study cannot be specific to any type of HF or CKD. However, the strong correlation between these pathways with HF and CKD proves the reliability and popularity of our results.To our knowledge, this study is the first study using the metaCCA method to identify shared risk genes and therapeutic targets of SGLT2 inhibitors associated with HF and CKD by effectively incorporating information from different GWAS datasets. Learning pleiotropic genes and their effects is essential. Compared to the animal experiments or one-trait GWAS, there are some advantages of our study. Firstly, the statistical power of the present study is higher than previous studies by integrating two large-scale GWAS summary statistics, which provided an increase in effective sample size up to 1 million. Secondly, the metaCCA method used published GWAS data, which does not require sequencing at the individual level, so it is a cost-effective method. Finally, with the help of the principle of the multitrait and multiomics, our study not only considered the internal correlation between genotypes but also the correlation between HF and CKD phenotypes . Using tIn the meantime, there are some limitations of this study. First of all, metaCCA mainly relies on statistical analysis, in which false positive results may occur. However, this may be addressed through the utilization of VEGAS2 analysis for validation of pleiotropic genes identified by metaCCA. It is still easy to have a particular SNP strongly correlated with multiple phenotypes making the gene or pathway in which the SNP is located meaningful . SecondlOur study performed a multivariate analysis by integrating VEGAS2 and TWAS to detect 169 pleiotropic genes. Moreover, we detected 21 therapeutic targets of SGLT2 that are associated with HF and CKD by integrating metaCCA and SGLT2 inhibitors' target prediction. In addition, we also illustrated the potential biological functions of these pleiotropic genes and therapeutic targets. Our results may provide novel insights into the shared genetic factors in the development of HF and CKD and common therapeutic targets of SGLT2 inhibitors shared by HF and CKD.


Some patients presented with EIMs before the diagnosis of IBD . Of these, seven cases (four in CD and three in UC) were visible by inspection of the patients (either dermatologic or ocular findings). The diagnosis of IBD and extraintestinal symptoms often occurred within a year (22.83% of CD and 16.04% of UC patients). Typically, the diagnosis of the first extraintestinal symptoms happened after the onset of bowel disease (+4.3 (±6.3) years, range: 10 years before to 30 years after in Crohn’s disease and +3.8 (±10) years, range: 24 years before to 30 years after) in ulcerative colitis. UC patients with pancolitis (E3) usually had EIMs earlier in the disease course and displayed EIMs more frequently before IBD diagnosis. Furthermore, patients with pancolitis developed EIMs more frequently than other sub-groups. Conclusion: Extraintestinal manifestations in inflammatory bowel diseases can present at any time, relative to the bowel symptoms. In cases, the presence of a characteristic EIM might be a harbinger of the development of IBD.Objectives: Thus far, few attempted to characterize the temporal onset of extraintestinal manifestations (EIM) in inflammatory bowel diseases (IBD). We sought to determine the time of onset of these findings in a patient cohort with IBD. Methods: We reviewed the electronic health records of 508 IBD patients  and summarized general patient characteristics and the temporal relationship and order of presentation of extraintestinal manifestations. Results: CD patients were younger at diagnosis. CD patients with ileocolonic involvement (L3) were younger, and UC patients with pancolitis (E3) were slightly younger at diagnosis. A total of 127 out of 303 (41.91%) CD and 81 out of 205 (39.51%) UC patients had EIMs ( Inflammatory bowel diseases (IBD: Crohn’s disease (CD) and ulcerative colitis (UC)) are chronic immune-inflammatory disorders, with predominantly gastrointestinal complaints ,2. CD caThus far, only Vavricka et al., assessed the temporal relationship of these conditions in adult IBD cases . While iIn our study, we sought to characterize patients with inflammatory bowel disease in Northeastern Hungary, treated by a single outpatient clinic, at the Clinical Center, University of Debrecen.We retrospectively searched the previous medical documentation of 508  patients with inflammatory bowel disease treated by a single outpatient clinic at the University of Debrecen Clinical Center. The collected data included gastroenterology visits from 2008 onwards until 2021. We included 133 males and 170 females with CD, whereas 89 males and 116 females had UC. Thus, the total number of male and female participants was 222 and 286, respectively. All of the patients had their condition confirmed via endoscopy and biopsy specimens.t-test was used to compare the two groups’ age of diagnosis. The recorded data included the age of diagnosis, the number of extraintestinal manifestations the patients had, and the temporal relationship  of these extraintestinal findings. In those with more than one extraintestinal manifestation, we also sought to organize these according to the sequence in which they appeared. Furthermore, Crohn’s disease patients were classified according to the Montréal system (to assess the length of inflamed bowel (L) and the behavior of their disease (B)). UC cases were evaluated by the maximum extent of colonic inflammation . We sought whether there were any characteristics of different Montréal E-groups . Comparison of three or more groups was carried out via ANOVA, and the interpretation of the results always considered the effect of the small size of our study cohorts.We collected the available data in an Excel spreadsheet . The extracted data were analyzed with Medcalc Software . Differences between the groups  were calculated. The comparison of proportions was completed through the chi-square test, as recommended for small sample sizes by Campbell . An inden = 303, 133 males, 170 females) were classified according to the Montréal system (age of disease onset (A), the length of inflamed bowel mucosa (L), disease behavior (B), and whether patients had perianal disease (p)). Montréal classification data are listed in p = 0.0313—data not shown). The most typical “age” category according to the Montréal system was A2 , diagnosed between 17 and 40 years of age).Crohn’s disease patients . We also included the age of diagnosis in UC with different Montréal E subtypes. For the results, please see The extent (length—L) of inflamed sections was also recorded. As we searched medical records from 2008 to 2021, the designated Montréal L and B subtypes reflected the disease course. The upper intestinal subtype, L4, was rare . L4 cases are probably being underreported, as upper endoscopy is not routinely performed during diagnostic workup . Upper gThe overview of extraintestinal manifestations is listed in p = 0.5898).Previous data are controversial as to whether EIMs are more common in CD or UC ,16,17,18n = 508 patients total; difference = 16.8 %, p = 0.0001). The difference between genders was not as marked when we compared the proportion of males and females with more than one EIM. In total, 9.9% of male and 15.38% of female patients had two or more EIMs, and this yielded a nonsignificant difference of 5.48%, p = 0.0686. This prevalence of multiple extraintestinal comorbidities is slightly less than we expected. In previous reports, more EIMs were present in roughly one-fourth of patients with IBD [The difference between males and females in whom EIMs were diagnosed was marked enough to reach a statistically significant level. In total, 70 male and 138 female patients had at least one EIM . Thereby, the percentage of affected patients was 31.53% in male and 48.25% in female patients , as well as axial skeleton involvement (sacroiliitis and spondylitis). Though peripheral arthritides are further subdivided into two types, we did not dichotomize, mostly due to insufficient available data. Axial joint complaints were equally common in CD and UC in our samples. Axial manifestations are reported to be more prevalent in males, but we could not confirm this. In this study sample, in total, 34 females and 17 males suffered from sacroiliitis, verified via imaging studies. The number of patients with sacroiliitis was greater in cases of CD , but this difference was nonremarkable. As the total number of patients was 508, of whom 51 had been diagnosed with sacroiliitis during their disease course, the prevalence of sacroiliitis was roughly 10% in our study group. This is lower, than we expected, as other studies reported greater prevalence ,23,24,25n = 10) or after the bowel disease onset (n = 13). Psoriasis—though an independent disease entity from IBD—was also found in five cases: two with CD and three with UC. It is noteworthy to add that two of those patients received biologic therapy  for their IBD; thereby, adverse drug effects (psoriasiform eruptions) cannot be ruled out in their cases [Skin lesions, specifically erythema nodosum (EN) and pyoderma gangrenosum (PG), are well recognized in IBD . Previouir cases .p = 0.4603; data not shown). This is contradictory to the findings reported in previously published studies [Comorbidities related to the hepatobiliary system were less prevalent. We did not register cases with liver steatosis (fatty liver) or hypertransaminasemia (elevated ALT and AST), as these conditions are common in the general population. Non-alcoholic fatty liver disease has an estimated global prevalence of 25%, this is similar to the incidences described in Hungary ,31. Chol studies ,32.p = 0.5761). This is also a conflicting finding with previous literature [The total number of cases with primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH) was low in our patient groups (the latter presenting exclusively in UC). Nevertheless, the two recorded cases with autoimmune hepatitis were diagnosed in patients also having primary sclerosing cholangitis. Both cases of AIH were recorded in male patients, even though this autoimmune disease is predominantly found in females ,34. Furtterature .We determined the proportion of patients in whom extraintestinal signs preceded the onset of IBD. We were not always able to determine the exact onset of these findings; thus, we only included cases wherein we had sufficient data on the disease course. The findings are listed in Even though certain visible-on-inspection extraintestinal findings might prove useful in assessing IBD risk, we found that these findings  seldom preceded the development of bowel disease. There were only seven cases total when either a dermatologic symptom or eye disease presented before IBD. Of these, three cases of erythema nodosum and one case of pyoderma gangrenosum were diagnosed in patients with Crohn’s disease, whereas there were two cases of iridocyclitis and one case of episcleritis in ulcerative colitis. Thus, Crohn’s disease patients were more inclined to develop dermatologic EIMs before their bowel symptoms. In ulcerative colitis, the preceding signs were more likely to be ophthalmologic (iridocyclitis and episcleritis). Nonetheless, the total number of cases and proportion of patients who developed EIMs before IBD was too small to draw any conclusions.p = 0.841), but this might have been overcome by a larger study group. . We thus conclude that a larger study group would have been needed to display marked differences between different groups.Furthermore, we sought to identify whether there were differences between different patient groups in the onset of EIMs according to the extent of their bowel disease. The overview of the results is provided in Moreover (data not shown), we found that in Crohn’s disease (when the onset of EIMs was determinable) with both ileal and colonic involvement , nearly 15% of patients had EIMs registered before the onset of IBD. This ratio was only 8% in Montréal L1 and L2 groups. Thus, it is possible that the presence of typical IBD-associated EIMs in otherwise non-symptomatic patients might foretell that later an extensive form of Crohn’s disease will develop—involving both the colon and ileum. A similar pattern was observable in ulcerative colitis as well. A total of 5 out of 16 patients (31%) with E3  developed EIMs before bowel symptoms, whereas this ratio was 6/24 (25%) in the E2 (left-sided colitis) and 3/30 (10%) in the E1 (proctitis) group. Although the samples were small, there was an observable tendency that greater affected bowel length meant an earlier diagnosis of an EIM.Another novel aspect of our results is that we attempted to organize the order of sequence in which EIMs appeared when patients suffered from more than one of these conditions. Thereby, we highlighted cases wherein more than one extraintestinal symptom was mentioned in the patient’s records. The data are summarized in p = 0.2130) supports the idea that patients are equally prone to developing further EIMs, regardless of whether they have CD or UC. Previous studies often reported multiple EIMs to be present in Crohn’s disease [p = 0.2033).We found that having more EIMs was equally common in CD and UC. Whereas 14.52% of all Crohn’s cases had two or more EIMs, this was 10.73% in UC patients . This 3.79% difference  occurred in 27 patients with Crohn’s disease and 15 patients with ulcerative colitis.In a considerable proportion of patients, the presentation of extraintestinal symptoms predated the onset of their bowel disease. We regard this to be an important point, as certain EIMs are visible upon inspection (such as ocular and dermatologic manifestations) and might raise the importance of being vigilant about subsequent bowel symptoms later. In our patient cohort, seven patients displayed “visible-on-inspection” extraintestinal findings. Of these, every dermatologic symptom was registered in patients who later developed Crohn’s disease, and ocular symptoms were seen in those who developed ulcerative colitis.We also aimed to determine whether there was a relationship between the affected bowel length and the presence of one or more EIM in Crohn’s disease. We found no difference between different Montréal L subgroups in this regard. We also evaluated whether there were differences in the onset of EIMs in different Montréal L subgroups, and we did observe a mild tendency that patients with colonic involvement had EIMs earlier in the disease course. In ulcerative colitis, the inflammation of the entire colon  predisposed patients to earlier development of EIMs. Moreover, roughly one-third of patients who developed pancolitis had their EIM registered before their bowel disease. We would like to mention that as we were collecting data across more years of patient history, we included the greatest extent of their bowel disease and classified patients accordingly. We did not register the severity of symptoms, while every patient underwent remission and occasional relapses during the disease course.In both CD and UC, the first extraintestinal symptoms were usually related to the musculoskeletal system and could affect both axial and peripheral joints. Notably, whenever more than one musculoskeletal EIM was present, patients usually developed peripheral arthritis earlier in the disease course.The dermatologic phenomenon known as metastatic CD is probably underreported . We wereAlthough not considered to be an extraintestinal manifestation of IBD, conjunctivitis and dry eye was the most frequently reported ophthalmologic condition in our patient cohort, and Sjögren’s syndrome was also diagnosed in seven patients total: four with Crohn’s disease and three with ulcerative colitis. Insufficient lacrimation is also recognized in IBD , thereby not every case with reduced tear production qualifies as Sjögren’s syndrome ,43. MoreThe management of the underlying IBD is crucial in most cases with EIMs. In modern era therapeutics, biologic agents  were shown to offer benefits during the course of various EIMs ,47,48,49The limitations of our paper stem from the design and execution: data were collected retrospectively and are thus prone to recall bias regarding the exact disease course of individual patients . We woulIn conclusion, extraintestinal manifestations are equally common in Crohn’s disease and ulcerative colitis, affecting roughly 4 in every 10 patients. Patients often experience more than one extraintestinal manifestation during the disease course. Though generally diagnosed after IBD onset, EIMs could also be present before bowel symptoms . Several patients had extraintestinal and bowel diseases recognized within a year (22.83% of CD and 16.04% of UC patients). Typically, the diagnosis of EIMs followed that of IBD, with +4.3 (± 6.3) years in Crohn’s disease and +3.8 (±10) years in ulcerative colitis. UC patients with more extensive inflammation (E3—pancolitis) usually developed EIMs earlier in the disease course, and there was a tendency in CD that the more extensive ileocolonic (L3) form also meant a somewhat earlier registration of EIM. Patients diagnosed at an earlier age were more likely to develop pancolitis in UC. The true prevalence of upper gastrointestinal lesions in CD is probably underreported.