The device-independent approach to physics is one where conclusions about physical systems (and hence of Nature) are drawn directly and solely from the observed correlations between measurement outcomes. This operational approach to physics arose as a byproduct of Bell’s seminal work to distinguish, via a Bell test, quantum correlations from the set of correlations allowed by local-hidden-variable theories. In practice, since one can only perform a finite number of experimental trials, deciding whether an empirical observation is compatible with some class of physical theories will have to be carried out via the task of hypothesis testing. In this paper, we show that the prediction-based-ratio method—initially developed for performing a hypothesis test of local-hidden-variable theories—can equally well be applied to test many other classes of physical theories, such as those constrained only by the nonsignaling principle, and those that are constrained to produce any of the outer approximation to the quantum set of correlations due to Navascués-Pironio-Acín. We numerically simulate Bell tests using hypothetical nonlocal sources of correlations to illustrate the applicability of the method in both the independent and identically distributed (i.i.d.) scenario and the non-i.i.d. scenario. As a further application, we demonstrate how this method allows us to unveil an apparent violation of the nonsignaling conditions in certain experimental data collected in a Bell test. This, in turn, highlights the importance of the randomization of measurement settings, as well as a consistency check of the nonsignaling conditions in a Bell test. In physics, the terminology “device-independent” apparently made its first appearance in Ref.  where thany local-hidden-variable (LHV) theory [A device-independent approach to physics, however, could be traced back, for example, to the work of Bell . There, ) theory  must be By now, this incompatibility has been verified in various loophole-free Bell tests, such as those reported in Refs. ,23,24,25p-value according to a certain null hypothesis . The corresponding p-value then describes the probability that the statistical model (associated with the null hypothesis) produces some quantity  at least as extreme as that observed.The effects of statistical fluctuations in a Bell test were  often reported in terms of the number of standard deviations the estimated Bell violation exceeds the corresponding local bound  [p-value upper bound—based on the data collected in a Bell test—according to LHV theories. As we shall show in this work, essentially the same method can be applied for the hypothesis testing of some other nonlocal physical theories, thus allowing us to bound the plausibility of physical theories beyond LHV theories.A pioneering work in this regard is that due to Gill  where het (CHSH)  Bell inet (CHSH)   ). This mIndeed, since the pioneering work by Popescu and Rohrlich , there hIn this work, we show that the prediction-based-ratio method can be applied to test any physical theory that is constrained to produce correlations that is amenable to a semidefinite programming  characteminimal assumptions. Making this possibility evident and demonstrating how well it works in practice are the main contributions of the present work.For a complete description of the prediction-based-ratio method and a comparison of its strength against the martingale-based method , we refex (y) and the corresponding measurement outcome (output) by a (b), where For simplicity, the following discussions are based on a Bell test that involves two parties (Alice and Bob) who are each allowed to perform one of two measurements randomly selected at each trial, each produces one of two possible outcomes. Generalization to other Bell scenarios will be evident. To this end, let us denote the measurement choice (input) of Alice (Bob) by In an LHV theory, the outcome probability distributions can be produced with the help of some LHV uch that :(1)PPA. Thelocality . A finerntum set , as it ax and y are to be chosen randomly according to some fixed probability distribution Coming back to an actual Bell test, let fixed state fixed measurement strategies If the experimental trials are independent and identically distributed (i.i.d.) corresponding to a Equation . In thisvergence   prediction-based-ratio method of Ref.  . It folg., Ref.  that thiThis Bell inequality is the key ingredient of the prediction-based-ratio method and is ideally suited for performing a hypothesis test of X and Y to denote the random inputs and the variables A and B to denote the random outputs of Alice and Bob at a trial. The ability to select measurement settings randomly, in particular, is an indispensable prerequisite of the prediction-based-ratio method, or more generally, a proper Bell test  as a non-negative function of the inputs To understand the method, we introduce the random variables g., Ref. ). We furtraining data and the data from the remaining hypothesis-testing data. In practice, we first construct the function R using the training data and then perform a hypothesis test with the test data. Since the ratio R is determined before the hypothesis test based on the prediction according to the training data, R is called a prediction-based ratio.Equation  is an alp-value. For concreteness, suppose that the actual measurements chosen at the i-th test trial are i-th test trial is T as the product of the possible values of the prediction-based ratio at all test trials, so the observed value of the test statistic is t can be expressed also asGiven a prediction-based ratio and a finite number p-value, which is defined as the maximum probability according to the hypothesis T at least as high as t actually observed in the experiment, is bounded byAccording to Ref. , the p-vp-value, the stronger the evidence against the hypothesis p-value bound computed in this manner remains valid even if the experimental trials are not i.i.d., while when the experimental trials are i.i.d., the p-value bound is asymptotically optimal (or tight) [The smaller the r tight) .p-value bound of Equation , it in Refs.  that EquOur second observation is that for the convex sets of correlations that are amenable to a semidefinite programming characterization, such as those considered in Refs. ,62,69,70Equation  is essenp-value hacking, it is essential that the test data used in the computation of the test statistic T is not used to determine R in Equation :=vP., Refs. ,57). In the CHSH  Bell ine“The observed data is compatible with a physical theory that is constrained to produce only the almost-quantum set of correlations.”with the same level of confidence. Inspired by the experiments of Ref. [To mimic an experimental scenario with noise (something unavoidable in practice), we shall introduce a slight perturbation to the ideal source Equation . Specifiin Refs. ,62.j-th extreme point of the nonsignaling polytope [Since we are interested to model a nonlocal source that obeys the nonsignaling conditions of Equation , there ipolytope , then weN-trial Bell test for any given input distribution Finally, to simulate the raw data by Minka .p-value upper bounds are summarized in Let us begin with the case of i.i.d. trials, corresponding to a source of correlation that remains unchanged throughout the experiment, and where the inputs at each trial are independent of the inputs of the previous trials. To this end, we first sample the weights p-value bounds obtained, 97% of them are trivial , while the smallest non-trivial p-value bound obtained is approximately 0.14. On the contrary, for the hypothesis test of the almost-quantum set of correlations, more than half of the simulated Bell tests give a p-value upper bound that is less than p-value bound according to the almost-quantum hypothesis, we see that the method generally works very well in falsifying this hypothesis. In fact, a separate calculation (not shown in the table) shows that when we increase p-value upper bounds obtained according to the almost-quantum hypothesis are less than or equal to As expected, despite statistical fluctuations, the data does not suggest any obvious evidence against the nonsignaling hypothesis. In fact, among the 500 i-th trial of the Bell test, we simulate according to the conditional outcome distributions:single nonsignaling extreme point used to mix with i-th trial, we randomly pick i-th trial) averages to that of Equation , however, turn out to be ttention  that durEquation , see, e.p-value bounds with fundamentally different features? The answer is negative. The reason is that in our crude application of the method, only the number of counts As discussed in the last section, the conclusion that “the experimental data of Ref.  show a vEquation . In partnon-i.i.d. experimental trials. Indeed, as we illustrate in p-value upper bound according to the nonsignaling hypothesis.So, if one holds the view that the nonsignaling principle cannot be flawed, then one must come to the conclusion that “should the measurement choices be randomized, it would be impossible to register the same number of counts p-value upper bound according to the nonsignaling hypothesis may be a consequence that certain premises needed to perform a sensible Bell test are violated. In other words, an apparent violation as such does not necessarily pose a problem to any physical principle, such as the nonsignaling principle that is rooted in the theory of relativity. However, as nonlocal correlations also find applications in device-independent quantum information processing [More generally, as the above example of ocessing ,19, it ip-value upper bound according to the nonsignaling hypothesis could also be a consequence of mere statistical fluctuation. Indeed, our results in true, it can still happen that one obtains a relatively small p-value upper bound  theory given the observed data, these results show that in most of these simulated Bell tests, the data is unlikely to be explicable by the assumed theory. In a similar manner, we numerically simulated another 500 Bell tests using a hypothetical source that varies from one trial to another. Again, the method remained very effective (giving a p-value upper bound that is less than To illustrate the effectiveness of the method, we first numerically simulated 500 Bell tests using a hypothetical source of correlations that lies somewhat outside the almost-quantum set of correlations. We then applied the method to obtain a p-value upper bounds (not randomized during the course of the experiments, thereby invalidating one of the basic assumptions needed in the application of the prediction-based-ratio method. Nonetheless, as we remarked in the Discussion section, the analysis nevertheless unveils that the possibility of using the prediction-based-ratio method to identify a situation where a certain premise is needed to perform a proper Bell test, such as the randomization of settings, is invalidated.Finally, we applied the prediction-based-ratio method to the experimental data of Ref. . To thisNote added: While preparing this manuscript, we became aware of the work of Smania et al. , which a


Eucalyptus globulus Labill.) and easy-  to-root species was carried out. Microcuttings from seedlings were inoculated in auxin-free AR induction medium and, after 96 h, transferred to AR formation medium for a period of 24 h. Samples were collected upon excision (Texc) and at the 5th day post excision (Tform). Delayed xylem development, with less lignification, was recorded in E. globulus, when compared to E. grandis, suggesting lower activity of the cambium layer, an important site for AR development. Superoxide was more densely present around the vascular cylinder at both sampled times, and in greater quantity in E. globulus than E. grandis, declining with time in the former. Hydrogen peroxide was localized primarily along cell walls, more intensely in the primary xylem and phloem, and increased significantly at Tform in E. globulus. Ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT) activities were generally higher in E. grandis and varied as a function of time in E. globulus. Soluble guaiacol peroxidase (GPRX) activity increased from Texc to Tform in both species, whereas cell wall-bound GPRX activity increased with time in E. grandis, surpassing E. globulus. Flavonoid content increased with time in E. grandis and was higher than E. globulus at Tform. Principal component analysis showed that species- and time-derived differences contributed to almost 80% of the variance. Overall, data indicate that E. grandis shows higher cambium activity and tighter modulation of redox conditions than E. globulus. These features may influence ROS-based signaling and phytohormone homeostasis of cuttings, thereby impacting on AR development. Besides being players in the realm of AR developmental differences, the specific features herein identified could become potential tools for early clone selection and AR modulation aiming at improved clonal propagation of this forest crop.Adventitious root (AR) development takes place in an intricate cellular environment. Reactive oxygen species (ROS) and antioxidant defenses, triggered by wounding in cuttings, can modulate this process. A comparative assessment of biochemical and anatomical parameters at critical rooting stages in hard- ( Adventitious roots (ARs) are formed post-embryonically from stems, leaves, hypocotyls, and other organs or tissues where previously there were no roots . AR deve2•−) into hydrogen peroxide (H2O2) and molecular oxygen , initially perceived only as toxic, have been shown to act as important signal transduction molecules in wound response . The balygen O2; . APX andelopment . APX useduce ROS . Flavonoduce ROS .2O2 might act as a signaling molecule, inducing AR formation  Microcutting anatomy features are related to root regeneration capacity; (ii) particular changes in ROS-related parameters of cuttings occur between the time of severance and early AR development; and (iii) distinct profiles of ROS-related parameters are apparent in eucalypt species of contrasting root regeneration capacity. The range of assessed parameters revealed specific patterns associated with each species and stage of AR development and may provide tools to characterize and possibly modulate rooting recalcitrance.Although it is known that ROS can play important roles in AR development, this oxidative component has not been examined in detail in Eucalyptus globulus Labill. and E. grandis W.Hill ex Maiden were used in the experiments. Seed surface sterilization and germination procedures were carried out according to −2 s−1 photosynthetically active radiation (provided by white LED lights) and temperature of 23 ± 2°C.in vitro adventitious rooting, 3-cm apical microcuttings with only two fully expanded leaf pairs were obtained by excision with a scalpel blade  to quantify rooting percentage, length of the longest root, and root number. For anatomical analysis and lignin staining, microcutting stem bases were fixed under vacuum in McDowell and Trump’s fixative solution of 4% formaldehyde and 1% glutaraldehyde in sodium phosphate buffer, pH 7.2  and 24 h after the transfer to formation medium . The latter harvest point was chosen as it generally coincides with early AR primordia development . For bio, pH 7.2 , and kepCross sections of 10–15 μm were obtained from fixed material using a handheld microtome. Histological sections were kept in distilled water to avoid dehydration and then transferred to spot plates for staining with phloroglucinol and HCl, according to −1 nitro blue tetrazolium (NBT), after which dark blue spots indicated NBT reduction. For H2O2 staining, separate sections were immersed in 1 mg ml−1 3,3'-diaminobenzidine (DAB) in 50 mM phosphate buffer (pH 7.0) for 30 min under LED lighting. Oxidized DAB produces a distinct brownish precipitate. For chlorophyll removal, sections were then transferred to 95% ethanol [v/v] for 15 min. Slides were prepared in water and images were obtained as described in Lignin staining section.Protocols for ROS visualization were adapted from n = 12) was defined as the area ratio of xylem tissue and the whole section. NBT- and DAB-stained area percentages were estimated using the Color Threshold feature of ImageJ2.All area measurements of images obtained through photomicrography were done using ImageJ2 v1.53c  and distn = 5) were ground in liquid nitrogen and solubilized in 1.5 ml extraction buffer . After centrifugation at 12,800 × g at 4°C for 15 min, the supernatant was recovered and immediately used for enzymatic activity assays. Protein content was determined according to Total protein extract was obtained from approximately 100 mg of frozen plant tissue. Samples (2O2) and 50 μl of the total protein extract (~56 μg protein). Changes in absorbance due to ascorbic acid oxidation  were monitored at 290 nm.Ascorbate peroxidase activity was assayed according to g was used for activity determination as described by 2O2), 20 μl of total extract (~22 μg protein) or cell wall-bound enriched extract were added. Tetraguaiacol formation  and 20 μl of total protein extract (~22 μg protein). H2O2 decomposition  were added. The reaction took place under white light for 15 min at room temperature. Final absorbance value was read at 560 nm. One SOD activity unit (U) was defined as the amount of active enzyme needed to inhibit NBT reduction by 50%.Superoxide dismutase activity was measured as per g for 10 min at 4°C and 100 μl of the supernatant were mixed with 400 μl of distilled water and 30 μl of 5% NaNO2 and kept at room temperature for 5 min. Then, 30 μl of 1% AlCl3 were added and vigorously mixed. After 6 min at room temperature, 200 μl of 1 M NaOH and 240 μl of distilled water were added and mixed well. The absorbance was measured at 510 nm and a standard curve was established using quercetin .Flavonoid content was estimated according to t-test using GraphPad Prism v8.0 for Windows , unless stated otherwise. Data were expressed as mean ± SEM and statistical significance was set at p ≤ 0.05.Means were compared by Student’s paired For principal component analysis (PCA), log-transformed data from three biological replicates for each species (sp.) × time permutation in respect to nine assessed variables were used. Factors were retained according to the Kaiser criterion, i.e., eigenvalue > 1. PCA was performed in R 4.0.3 , using pE. grandis showed higher rooting capacity when compared to E. globulus  to obtain more uniform microcuttings. To ensure that differences in xylem area percentage were not a matter of growth, 16-week E. globulus microcuttings were also assessed. Mean xylem area percentage was equal to 14-week E. globulus and inferior to 16-week E. grandis  stand amid the various quantitative parameters, and if the overall picture may be associated with the divergent rooting capacity, a PCA was performed. Nine variables, i.e., xylem percentage area (Xylem%), DAB-stained percentage area (DAB-area), and NBT-stained percentage area (NBT-area) in histological sections, SOD, APX, CAT, soluble GPRX (SolGPRX), cell wall-bound GPRX (CWGPRX) activities, and flavonoid content (flav) in microcuttings were used in the analysis.2 values , the contribution (%) of each variable to each PC can be estimated , “SolGPRX” showed strong negative correlation with PC1  was explained by PC1  and PC2 (37.8% – associated with the “time” factor), a notable 13% constituted sp. × time factor interaction-derived variance. Out of the nine parameters entered in the PCA, NBT-stained area percentage almost single-handedly represented the interaction.E. grandis microcuttings when compared to E. globulus , an auxin-response module related to wood formation, led to lower xylem area percentages  has been associated with this phenomenon, since r extent .Arabidopsis. Also essential to ROS propagation and proper signaling were cell wall-bound peroxidases of hypocotyls  of differently abundant proteins to be redox-related. Patterns also reflected changes in proteomic profiles from the induction to the formation stage of AR development (Soluble peroxidase activity increased with age in  content . Cucumbe content . Solublecentages . In the elopment .transparent testa 3 (tt3) Arabidopsis mutants, which accumulate flavonols, whereas flavonoid-deficient tt4 mutants had higher concentrations of the same catabolite. This indicates a possible role for flavonoids as ROS-induced auxin degradation buffers (E. gunnii cutting flavonoid content has been associated with overcoming of AR recalcitrance (Nonenzymatic antioxidants have also been implicated in auxin-ROS crosstalk. Among these, flavonoids stand out for their multiple interactions with factors that play roles in this scenario. These molecules are known to interfere with auxin transport and thought to mitigate local ROS accumulation that could hinder auxin homeostasis . The mai buffers . In a co buffers . An incrcitrance . In the Eucalyptus spp. adventitious rooting, the data from this study revealed different profiles, explained mainly by species- and time-linked variations. Eucalyptus grandis microcuttings showed larger xylem area than E. globulus. Along with this feature, SOD and both cell-wall bound and soluble GPRX activities were indicative of rooting capacity. DAB-stained area, as well as APX and CAT activities, were associated with changes from cutting excision to the AR formation stage, regardless of species. Flavonoid content was associated with both time- and species-derived variation. Overall, data indicate that E. grandis shows higher cambium activity and tighter modulation of redox conditions than E. globulus. These features may influence ROS-based signaling and phytohormone homeostasis of cuttings, thereby impacting on AR development. Besides being players in the realm of AR developmental differences, the specific features herein identified could become potential tools for early clone selection and AR modulation aiming at improved clonal propagation of this forest crop.As a first incursion into the complexity of the comparative redox status during Eucalyptus spp. and their hybrids across the spectrum of rooting capacity. If validated under commercial greenhouse production settings and across several genotypes, some of the parameters explored in this study may contribute to the establishment of robust global models for early prediction of rooting recalcitrance, thereby contributing to germplasm selection and clonal propagation programs.Future studies of AR recalcitrance will be able to further explore parameters that correlate with rooting capacity and add elements such as phytohormones and additional antioxidants, as well as other The original contributions presented in the study are included in the article/in vitro culture system and performed biochemical analyses. JV, CC, and JM sectioned, stained, and imaged microcuttings. JV performed the statistical analysis. CC and JV wrote the first draft of the manuscript. AF-N reviewed and finalized the manuscript, and warranted financial support. All authors contributed to manuscript revision, read, and approved the submitted version.JV, CC, and AF-N contributed to conception and design of the study. JV, CC, and LR set up the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Syncephalastrum racemosum (RCMB 05922) in comparison to the reference drugs, while compounds 4b and 4h demonstrated the highest inhibitory activity against Escherichia coli (E. coli) in evaluation against the reference drugs. Moreover, their cytotoxicity was assessed against three different human cell lines, including human colon carcinoma (HCT-116), human hepatocellular carcinoma (HepG-2), and human breast adenocarcinoma (MCF-7) with a selection of molecules illustrating potency against the HCT-116 and MCF-7 cell lines. Furthermore, the molecular modeling results depicted the binding interactions of the synthesized compounds 3b and 3h in the active site of the E. coli DNA gyrase B enzyme with a clear SAR (structure–activity relationship) analysis. Lastly, the density functional theory’s (DFTs) theoretical calculations were performed to quantify the energy levels of the Frontier Molecular Orbitals (FMOs) and their energy gaps, dipole moments, and molecular electrostatic potentials. These data were utilized in the chemical descriptor estimations to confirm the biological activity.The high biological activity of the chromene compounds coupled with the intriguing optical features of azo chromophores prompted our desire to construct novel derivatives of chromene incorporating azo moieties 4a-l, which have been prepared via a three-component reaction of 1-naphthalenol-4-[(4-ethoxyphenyl) azo], 1, with the benzaldehyde derivatives and malononitrile. The structural identities of the azo-chromene 4a-l were confirmed on the basis of their spectral data and elemental analysis, and a UV–visible study was performed in a Dimethylformamide (DMF) solution for these molecules. Additionally, the antimicrobial activity was investigated against four human pathogens (Gram-positive and Gram-negative bacteria) and four fungi, employing an agar well diffusion method, with their minimum inhibitory concentrations being reported. Molecules 4a, 4g, and 4h were discovered to be more efficacious against  The last century has seen an exponential increase in the growth of cancerous masses and this has emerged as one of the primary global causes of mortality in this age. Due to the presence of genetic and environmental factors such as mutations, carcinogens, detrimental nutrition, and emissions, cancer incidence has been on the rise within both developing and developed countries . To combA suitable candidate of these criteria is the family of chromene molecules, which have captivated cancer researchers for their diversified and assorted medicinal applications, specifically anti-inflammatory, anti-cancer, and anti-fungal ,6,7,8,9.H-chromenes inspire many researchers to synthesize new compounds featuring different aromatic rings fused to the chromene moiety and obtain more active derivatives, as they are reliant upon the pattern of substitution. Of these aromatic derivatives, azo chromophores have been utilized due to their comprehensive applications in analytical chemistry chromene-3-carbonitrile derivatives in good yield, as illustrated in In our attempts to synthesize chromene derivatives, the three-component condensation reaction of 1-naphthalenol-4-[(4-ethoxyphenyl) azo] The aforementioned Knoevenagal condensation, followed by the Michael addition adducts, is the fundamental methodology for this class of synthesis, and the plausible mechanism  is repre4a-l were confirmed with the aid of spectroscopic data. Case in point, the FT-IR spectroscopy of the acquired compounds revealed characteristic absorption bands between 2187 and 2210 for the CN groups while the NH2 stretches were in the range of 3336–3470 cm−1. Additionally, the 1H NMR spectra of the chromene compounds 4a-l were obtained in DMSO-d6. As anticipated, the methyl protons appeared as a triplet set at 1.34–1.37 ppm with the H-4 pyran displaying signals at 5.07–5.49 ppm and the amine protons resonating at 6.71–7.11 ppm. Meanwhile, the singlet at 4.11–4.19 ppm corresponds to the methylene group. The rest of the aromatic protons resonate further downfield in the range of 6.62–8.97 ppm. The 13C NMR spectra of the synthesized molecules revealed the carbon atoms’ attachment to the methyl protons, resonating at 15.40–14.48 ppm, while the methylene carbons appeared at 64.58–64.14 ppm. In addition, the signal at 41.71–31.52 ppm corresponded to the C-4 of the pyran ring, and the quaternary carbon C-2, which is attached to the amine group, appeared in the range of 58.49–55.67 ppm. Lastly, the CN carbon resonated further downfield at 118.92–117.69 ppm, and the aromatic CH carbons exhibited signals between 144.06 and 111.75 ppm.The structures of these chromene compounds 4(a-j) were dissolved in a Dimethylformamide (DMF) solution with a concentration of 10−3 M in order to examine their UV–Vis absorption spectroscopy. The spectra of all examined compounds demonstrated two comparable peaks around 393 and 591 nm. The first band appeared due to the n-π* transition with a substantial charge-transfer feature expressed in the peak’s broadness. Meanwhile, the longer wavelength band is attributed to the weak forbidden n-π*, the π-π*, and the solvated complex development via an intermolecular H-bonding between the chromene dyes and the DMF, The new highly colored molecules 4a-j exerted a bathochromic shift in their absorption maxima, owing to their protonation and the establishment of the azonium configuration. 4a upon the addition of an acid, evidenced by the appearance of two longer λmax values at 524 and 408 nm chromen-6-yl)diazene diazene were visualized using BIOVIA Discovery studio visualizer.The molecular docking calculations of the synthesized derivatives bacteria . Meanwhioftwares ,53. To voftwares . The Fatn group,  were uti4b and 4h in the pocket is explained through three different fragments around the chromene scaffold: 8-NH2, 9-CN, and -Br, which belong to 4h. These fragments formed stable hydrogen bonding interactions with a panel of corresponding pocket residues: Asp 73, Thr 165, Asn 46, Asp 49, and Ile 78 with different distances between 2.00 and 3.23 Å. Fat Brown B displayed diverse interactions, including a hydrogen bonding interaction with Asp 73 (2.45 Å), while no hydrogen bond was established with 1-(4-ethoxyphenyl)-2-(4-phenyl-4H-benzo[h]chromen-6-yl)diazene, and this might be the reason for their lower binding affinity with the target receptor. This proves the importance of the synthesized compounds bearing NH2, CN, COOC2H5, and halogen group over the starting material, i.e., Fat Brown B and 1-(4-ethoxyphenyl)-2-(4-phenyl-4H-benzo[h]chromen-6-yl)diazene , The results of the docking experiments were summarized in It is well documented that the energy values of the HOMOs and LUMOs for a specific molecule can be used as a qualitative tool to describe the molecule’s ability to donate or receive electrons from a receptor molecule ,57,58. TGlobal hardness (η) could be used to predict a number of thermodynamic properties of the molecule including polar surface area, ovality, hydrophobicity, volume, electronegativity (χ), dipole moment (µ), and the level of charge transfer prevention of the molecule.ω) could be calculated from the values of the electronegativity and chemical hardness and were also calculated from the energy levels of the FMOs, as seen in 4a-l is shown in 4g, exhibiting the lowest energy level and 4l the highest-lying level. On the other hand, the level of the LUMOs fluctuated between −3.1 eV to −2.4 eV with 4g, demonstrating the high-lying LUMO and compound 4l at the low-lying level. Therefore, molecule 4g is the most capable of accepting electrons from neighboring receptors, and compound 4l is the most capable of donating electrons to neighboring acceptors. The energy gaps between the FMO levels of the prepared compounds were in the range of 2.9 to 3.3 eV. It is clear that molecules 4a and 4g in comparison with the other molecules possess the highest and the lowest energy difference, respectively, and this could illustrate their high binding affinity in terms of the molecular docking scores for the measured biological activity.Furthermore, the electrophilicity  between such proposed compounds suggested a preference of 4k and 4i rather than 4h for enzyme inhibition. Furthermore, compounds 4h and 4l demonstrated the highest H-bonding either in donation or the polar surface area with respect to the other compounds, Alternatively, the calculated lipophilicity revealed the ability of the drug-like molecules to flux in lipophilic parts, which allows passage through several membranes of the biological organs and results in cytotoxic molecules. The variation in the calculated log P  is an important parameter to be predicted since the MEP is a sign about the molecular size and shape of the positive, negative and the neutral electrostatic potential, and can be a tool for expecting physiochemical property relationships with the molecular structure. Furthermore, the molecular electrostatic potential is a useful tool in the prediction of the susceptibility of the studied compounds towards electrophiles and nucleophiles.. In the MEP, the higher negative region is the favored site for electrophilic attack shown in the red color. So, an attacking electrophile will be attracted by the negatively charged sites and the opposite situation for the blue regions. It is obvious that the molecular size and the shape as well as the orientation of the negative, positive, and the neutral electrostatic potential are varied according to the electronic nature of the compound as well as the electronegativity of the attached groups. The difference in the mapping of the electrostatic potential around the compounds could be responsible for the extent of binding affinity of the studied compounds to the active site of the receptor.The molecular electrostatic potential (MEP) was calculated by the same method under the same base sets as the previous calculations, Figure 111 was purchased from Sigma-Aldrich . Melting points were determined in open capillaries using an electrothermal apparatus  and are uncorrected. The progress of the reactions was monitored using thin-layer chromatography (TLC) on Merck silica gel 60 F254 plates. Infrared (IR) spectra were recorded using Bruker Alpha FT-IR Spectrometer  as pressed KBr pellets. 1H-NMR and 13C-NMR spectra were recorded on 300 MHz and at 75 MHz, respectively, on a Bruker Avance Spectrometer  in DMSO-d6 with tetramethylsilane as an internal standard. The elemental analyses for C, H, and N were performed using an Exeter Analytical, Inc. CE-440 Elemental Analyser . UV–vis absorption measurements were performed using a HP8543 UV–vis spectrophotometer.Chemicals and solvents were purchased from Sigma-Aldrich  and Alfa Aesar  and were used as received. Compound The microorganism inoculums were uniformly spread using sterile cotton swabs on a sterile Petri dish malt extract agar (for fungi) and nutrient agar (for bacteria). One hundred cubic millimeters of each sample were added to each well . The systems were incubated for 24–48 h at 37 °C (for bacteria) and at 28 °C (for fungi). After incubation, microorganism growth was observed. Inhibition zones of the bacterial and fungal growth were measured in millimeters. Tests were performed in triplicate ,81.2 and then washed with sterile PBS . The cells were then treated with 100 µL from different dilutions of tested sample in a fresh maintenance medium and incubated at 37 °C. Negative control samples were left untreated and positive controls containing doxorubicin were also evaluated for comparison as a reference drug. For each concentration of each test sample, six wells were used and observation was conducted every 24 h using an inverted microscope. The surviving cells were stained with crystal violet followed by cell lysing using 33% glacial acetic acid and then mixed. The absorbance at 590 nm using a microplate reader  was measured and the absorbance values from untreated cells were considered as 100% proliferation [50) was estimated from the graph.All cell lines including HCT-116 (human colon carcinoma), HepG-2 (human hepatocellular carcinoma), and MCF-7 (human breast adenocarcinoma) were received from the American Type Culture Collection . The cells were grown as monolayers on RPMI-1640 medium  supplemented with 50 µg/mL gentamycin and 10% inactivated FCS . The potential cytotoxicity of the target compounds was evaluated using the method published by Gangadevi and Muthumary . The celferation ,83. UsinE. coli topoisomerase II DNA gyrase B (PDB code 1KZN). The MOE software [The newly synthesized compounds were docked into the crystal structure of software  was usedThe theoretical calculations for the investigated compounds were carried out by Gaussian 09 software . DFT/B3LYP methods using 6-31G  basis set was selected for the calculations. The geometries were optimized by minimizing the energies with respect to all geometrical parameters without imposing any molecular symmetry constraints. The structures of the optimized geometries had been drawn with Gauss View. Moreover, the calculated frequencies were carried out using the same level of theory. The frequency calculations showed that all structures were stationary points in the geometry optimization method with none imaginary frequency.4a-l.A mixture of compound 1 (2.3 mmol), malononitrile or ethyl cyanoacetate (2.3 mmol), and aryl aldehyde (2.3 mmol) in ethanol (5 mL) with a few drops of pipredine was added. The reaction mixture was stirred at reflux. After completion of the reaction (monitored by TLC), the mixture was cooled and filtered, washed with ethanol and hexane to afford −1: 3469, 3280 (NH2), 2925, 2891 (CH), 2201 (CN), 1571 (N = N); 1H NMR  δ 8.87–8.83 , 8.39–8.34 , 7.94 , 7.81–7.77 , 7.41 , 7.37–7.28 , 7.22–7.14 , 7.10 , 5.27 , 4.14 , 1.36 ; 13C NMR  δ 162.30 (C-2), 160.96, 147.53, 146.07, 143.96, 132.72 (Ar-C), 132.55, 131.35 (Ar-CH), 131.15 (Ar-C), 130.35, 128.81 (Ar-CH), 128.32 (Ar-C), 125.76, 124.11, 121.98, 120.85 (Ar-CH), 117.80 (CN), 116.95 (Ar-C), 116.67, 115.90, 112.18 (Ar-CH), 64.57 (CH2), 55.67 (C-3), 31.52 (CH-4), 15.39 (CH3); C28H21N4O2F; Calculated: C, 72.40; H, 4.56; N, 12.06. Found: C, 73.05; H, 4.50; N, 11.87.Brown solid (85%), m.p. 205 °C; IR (KBr) cm−1: 3466, 3326 (NH2), 2995, 2973, 2932 (CH), 2197 (CN), 1567 (N = N); 1H NMR  δ 8.81-8.79 , 8.37-8.34 , 7.90 , 7.78–7.75 , 7.45 , 7.40-7.21 , 7.07 , 5.46 , 4.11 , 1.35 ; 13C NMR  δ 162.32 (C-2), 160.86, 147.49, 146.03, 143.92, 142.72 (Ar-C), 133.01, 132.22, 131.38 (Ar-CH), 130.93 (Ar-C), 129.98, 128.87, 128.35 (Ar-CH), 125.78 (Ar-C), 124.07, 123.97, 122.03, 120.71 (Ar-CH), 117.69 (CN), 115.91 (Ar-CH), 111.87 (Ar-C), 64.57 (CH2), 55.78 (C-3), 31.53 (CH-4) 15.39 (CH3); C28H21N4O2Cl; Calculated: C, 69.92; H, 4.40; N, 11.65. Found: C, 71.35; H, 4.02; N, 11.75.Greenish-brown solid (87%), m.p. 219 °C; IR (KBr) cm−1: 3468, 3326 (NH2), 2995, 2973, 2932 (CH), 2198 (CN), 1569 (N = N); 1H NMR  δ 8.86–8.80 , 8.39–8.34 , 7.92 , 7.80–7.76 , 7.64 , 7.40–7.27 , 7.25–7.19 , 7.09 , 5.49 , 4.13 , 1.36 ; 13C NMR  δ 162.32 (C-2), 160.78, 147.46, 145.89, 143.91 (Ar-C), 134.10, 132.45, 131.39, 130.24 (Ar-CH), 129.54 (Ar-C), 128.86, 128.34, 125.78 (Ar-CH), 124.11(Ar-C), 123.96 (Ar-CH), 123.32 (Ar-C), 122.06, 120.65 (Ar-CH), 117.80 (CN), 115.89 (Ar-CH), 111.75 (Ar-C), 64.56 (CH2), 56.01 (C-3), 31.53 (CH-4), 15.38 (CH3); C28H21N4O2Br; Calculated: C, 64.01; H, 4.03; N, 10.66. Found: C, 65.78; H, 3.67; N, 10.78.Brown solid (83%), m.p. 208 °C; IR (KBr) cm−1: 3466, 3335 (NH2), 2975, 2935, 2883 (CH), 2193 (CN), 1566 (N = N); 1H NMR  δ 8.89–8.80 , 8.37–8.36 , 7.95 , 7.81-7.77 , 7.50 , 7.46-7.42 , 7.34–7.29 , 7.12 , 5.09 , 4.15 , 1.37 ; 13C NMR  δ162.33 (C-2), 160.68, 149.15, 147.55, 145.82 (Ar-C), 144.06, 132.04, 131.33 (Ar-CH), 131.20 (Ar-C), 130.94, 128.88, 128.36 (Ar-CH), 127.83 (Ar-C), 125.78, 124.22 (Ar-CH), 122.89 (Ar-C), 122.05, 120.89 (Ar-CH), 118.38 (CN), 115.93 (Ar-CH), 112.48 (Ar-C), 64.58 (CH2), 56.89 (C-3), 31.53 (CH-4), 15.40 (CH3); C28H21N4O2Br; Calculated: C, 64.01; H, 4.03; N, 10.66. Found: C, 66.27; H, 3.77; N, 10.87.Brown solid (86%), m.p. 217 °C; IR (KBr) cm−1: 3470, 3336 (NH2), 2979, 2927, 2875 (CH), 2194 (CN), 1566 (N = N); 1H NMR  δ 8.97-8.90 , 8.44-8.38 , 7.98 , 7.80–7.73 , 7.55 , 7.43-7.27 , 7.15 , 7.12 , 6.62 , 5.11 , 4.19 , 1.43 ; 13C NMR  δ 162.40 (C-2), 148.39, 147.68, 144.49, 134.54, 131.60 (Ar-C), 130.95, 128.32 (Ar-CH), 128.10 (Ar-C), 127.70, 127.05, 125.34 (Ar-CH), 125.16 (Ar-C), 124.34, 123.77, 122.49, 121.60, 119.23 (Ar-CH), 117.95 (CN), 115.28 (C-Ar-CH), 112.22 (Ar-C), 64.14 (CH2), 58.49 (C-3), 41.71 (CH-4), 14.48 (CH3); C28H21N4O2Cl; Calculated: C, 69.92; H, 4.40; N, 11.65. Found: C, 70.17; H, 4.21; N, 11.70.Dark brown solid (88%), m.p. 212 °C; IR (KBr) cm−1: 3466, 3278 (NH2), 2979, 2935, 2869 (CH), 2187 (CN), 1567 (N = N); 1H NMR  δ 8.86–8.81 , 8.38–8.35 , 7.95 , 7.81–7.76 , 7.41 , 7.35–7.27 , 7.19–7.09 , 5.07 , 4.15 , 1.37 ; 13C NMR  δ 162.31(C-2), 160.51, 147.54, 145.74, 143.99, 142.69, 131.26 (Ar-C), 130.59, 130.48 (Ar-CH), 128.81 (Ar-C), 128.32, 125.76, 125.24 (Ar-CH), 124.21 (Ar-C), 123.98, 122.02 (Ar-CH), 120.99 (Ar-C), 118.92 (CN), 116.61, 116.32, 115.92, 112.60 (Ar-CH), 64.57 (CH2), 57.34 (C-3), 31.54 (CH-4), 15.40 (CH3); C28H21N4O2F; Calculated: C, 72.40; H, 4.56; N, 12.06. Found: C, 71.36; H, 4.32; N, 11.78.Greenish-brown solid (84%), m.p. 214 °C; IR (KBr) cm−1: 3418, 3331 (NH2), 2980, 2967, 2923 (CH), 2210 (CN), 1571 (N = N), 1473, 1352 (NO2); 1H NMR  δ 8.85–8.81 , 8.41–8.37 , 8.18 , 8.12 , 7.92 , 7.82–7.76 , 7.64 , 7.45-7.41 , 7.09 , 5.32 , 4.12 , 1.35 ; 13C NMR  δ 162.33 (C-2), 160.83, 148.91, 148.58, 147.50, 145.91, 144.16 (Ar-C), 135.47, 131.47 (Ar-CH), 131.39 (Ar-C), 128.96, 128.40, 125.75 (Ar-CH), 124.23 (Ar-C), 124.02, 123.14, 122.98 (Ar-CH), 122.07 (Ar-C), 120.80 (Ar-CH), 117.99 (CN), 115.89, 112.43 (Ar-CH), 64.56 (CH2), 56.56 (C-3), 31.52 (CH-4), 15.38 (CH3); C28H21N5O4; Calculated: C, 68.42; H, 4.31; N, 14.25. Found: C, 69.33; H, 4.20; N, 13.46.Brown solid (88%), m.p. 216 °C; IR (KBr) cm−1: 3451, 3296 (NH2), 2981, 2939, 2885 (CH), 1674 (CO), 1516 (N = N); 1H NMR  δ 8.80 , 8.42 , 7.91 , 7.79–7.71 , 7.67 , 7.56 , 7.27–7.19 , 7.11-7.05 , 5.64 , 4.12 , 3.51 , 1.36 ; 13C NMR  δ 169.22 (C=O), 168.92, 162.25 (Ar-C), 161.22 (C-2), 147.61, 147.47, 145.45 (Ar-C), 143.94, 133.50, 131.21, 129.24 (Ar-CH), 129.12 (Ar-C), 128.58, 128.15, 125.72 (Ar-CH), 124.20 (Ar-C), 123.16 (Ar-CH), 122.96 (Ar-C), 122.07 (Ar-CH), 120.94 (Ar-C), 115.89, 111.92 (Ar-CH), 76.29 (C-3), 64.55 (CH2), 51.36 (OCH3), 28.16 (CH-4), 15.38 (CH3); C29H24N3O4Br; Calculated: C, 62.94; H, 4.58; N, 7.34. Found: C, 63.75; H, 3.85; N, 8.10.Dark brown solid (83%), m.p. 145.2 °C; IR (KBr) cm−1: 3454, 3316 (NH2), 2977, 2934 (CH), 1675 (CO), 1535 (N = N); 1H NMR  δ 8.79-8.76 , 8.41–8.37 , 7.93–7.86 , 7.75-7.67 , 7.56 , 7.37 , 7.26 , 7.20 , 7.13 , 7.05 , 5.60 , 4.08 , 3.52 , 1.33 ; 13C NMR  δ 207.36 (C = O), 169.18, 168.88, 162.24 (Ar-C), 161.36 (C-2), 147.49, 145.54, 143.93 (Ar-C), 132.27, 131.24, 131.19 (Ar-CH), 130.33 (Ar-C), 128.88, 128.58, 128.16, 125.81 (Ar-CH), 124.14 (Ar-C), 123.89, 122.05 (Ar-CH), 120.71 (Ar-C), 115.89, 112.05 (Ar-CH), 75.97 (C-3), 64.55 (CH2), 51.38 (OCH3), 32.97 (CH-4), 15.38 (CH3); C29H24N3O4Cl; Calculated: C, 68.24; H, 4.96; N, 7.96. Found: C, 68.91; H, 4.20; N, 8.69.Light brown solid (81%), m.p. 139.9-140 °C; IR (KBr) cm−1: 3489, 3346 (NH2), 2976, 2949, 2871 (CH), 1675 (CO), 1487 (N = N); 1H NMR  δ 8.82 , 8.43 , 7.95 , 7.89 , 7.80–7.71 , 7.60 , 7.43 , 7.33-7.26 , 7.22-7.18 , 7.11 , 5.16 , 4.13 , 3.57 , 1.36 ; 13C NMR  δ 169.05 (C = O), 162.25 (Ar-C), 161.31 (C-2), 151.13, 147.57, 145.96, 144.14 (Ar-C), 131.64 (Ar-CH), 131.12 (Ar-C), 130.73, 130.08, 128.60, 128.18, 127.35, 125.72 (Ar-CH), 124.19 (Ar-C), 123.97 (Ar-CH), 122.51 (Ar-C), 122.05 (Ar-CH), 121.46 (Ar-C), 115.88, 112.89 (Ar-CH), 79.93 (C-3), 78.32 (CH2), 57.67 (OCH3), 39.95 (CH-4), 18.45 (CH3); C29H24N3O4Br; Calculated: C, 62.94; H, 4.58; N, 7.34. Found: C, 63.20; H, 3.90; N, 7.74.Orange solid (86%), m.p. 158.9 °C; IR (KBr) cm−1: 3489, 3346 (NH2), 2976, 2949, 2871 (CH), 1675 (CO), 1487 (N = N); 1H NMR  δ 8.82 , 8.43 , 7.95 , 7.89 , 7.80–7.71 , 7.60 , 7.43 , 7.33-7.26 , 7.22-7.18 , 7.11 , 5.16 , 4.13 , 3.57 , 1.36 ; 13C NMR  δ 169.05 (C = O), 162.25 (Ar-C), 161.31 (C-2), 151.13, 147.57, 145.96, 144.14 (Ar-C), 131.64 (Ar-CH), 131.12 (Ar-C), 130.73, 130.08, 128.60, 128.18, 127.35, 125.72 (Ar-CH), 124.19 (Ar-C), 123.97 (Ar-CH), 122.51 (Ar-C), 122.05 (Ar-CH), 121.46 (Ar-C), 115.88, 112.89 (Ar-CH), 79.93 (C-3), 78.32 (CH2), 57.67 (OCH3), 39.95 (CH-4), 18.45 (CH3); C29H24N3O4Br; Calculated: C, 62.94; H, 4.58; N, 7.34. Found: C, 63.20; H, 3.90; N, 7.74.Orange solid (85%), m.p. 158.9 °C; IR (KBr) cm−1: 3466, 3278 (NH2), 2979, 2935, 2869 (CH), 2187 (CN), 1567 (N = N); 1H NMR  δ 8.86–8.81 , 8.38–8.35 , 7.95 , 7.81–7.76 , 7.41 , 7.35–7.27 , 7.19–7.09 , 5.07 , 4.15 , 1.37 ; 13C NMR  δ 162.31(C-2), 160.51, 147.54, 145.74, 143.99, 142.69, 131.26 (Ar-C), 130.59, 130.48 (Ar-CH), 128.81 (Ar-C), 128.32, 125.76, 125.24 (Ar-CH), 124.21 (Ar-C), 123.98, 122.02 (Ar-CH), 120.99 (Ar-C), 118.92 (CN), 116.61, 116.32, 115.92, 112.60 (Ar-CH), 64.57 (CH2), 57.34 (C-3), 31.54 (CH-4), 15.40 (CH3); C28H21N4O2F; Calculated: C, 72.40; H, 4.56; N, 12.06. Found: C, 71.36; H, 4.32; N, 11.78.Greenish-brown solid (88%), m.p. 214 °C; IR (KBr) cm4a-l via the Knoevenagal condensation of the benzaldehyde derivatives and malononitrile or ethyl cyanoacetate, followed by the electrophilic substitution step of 1-naphthalenol-4-[(4-ethoxyphenyl) azo (Michael addition adduct). All the newly synthesized compounds were characterized, utilizing IR and NMR spectroscopy. The antimicrobial activity of the desired derivatives illustrated greater potency in appraisal with the reference drugs against certain fungal species and Gram-negative bacteria. On the other hand, these molecules, in evaluation with the same references, revealed comparable activity towards Gram-positive bacteria. Additionally, the novel compounds witnessed comparable or slightly lower cytotoxicity in comparison with the reference compounds. Overall, compounds 4l, 4j, and 4e displayed good IC50, ranging from 4.35 to 5.54 µg/mL, against the HCT-116 with compounds 4l, 4j, and 4h, exhibiting strong behavior against the MCF-7 cell lines. Moreover, all the target derivatives revealed less activity in the case of HepG-2 cell line except 4l. The docking study results demonstrated the binding interactions of the synthesized compounds 4b and 4h in the active site of the E. coli DNA gyrase B enzyme. Lastly, the DFT results in terms of the structure–activity relationships (SAR) were also constructed to validate the obtained findings of the prepared compounds’ biological activity. Furthermore, the density distributions of the FMOs surfaces were employed to describe the molecules’ different biological activity profiles, where the active compounds HOMO’s character exhibited less density than their LUMO’s character, while the symmetrical distribution of these orbitals is related to lower biological activity.This report discussed the synthesis of novel highly-colored chromene compounds incorporating azo chromophore moieties


Aortic regurgitation (AR) was recognized as a major, but rare complication after device closure for perimembranous ventricular septal defects (PmVSD). Most of them are temporary and non-significant. Infectious endocarditis (IE) is another extremely rare post-procedure complication of PmVSD. Theoretically, AR could increase risk for post-interventional IE. However, no cases have been documented thus far. We firstly described a case of very late-onset IE associated with non-significant AR after transcatheter closure of PmVSD with modified symmetrical double-disk device, underscoring the need for reassessing long-term prognostic implications of non-significant post-procedure AR after PmVSD occlusion and the most appropriate treatment strategy.A 15-year old male received transcatheter closure of a 6.4 mm sized PmVSD with a 9-mm modified symmetric double-disk occluder (SHAMA) 11 years ago in our hospital. A new-onset mild eccentric AR was noted on transthoracic echocardiography (TTE) examination 1-year post procedure, without progression and heart enlargement. At this time, the child was admitted with a complaint of persistent fever for 16 days and nonresponse to 2-weeks course of amoxicillin and cefoxitin.Staphylococcus aureus from all three-blood cultures.The diagnosis of post-procedure IE was established since a vegetation (14 × 4 mm) was found to be attached to the tricuspid valve, an anechoic area (8 × 7 mm) on left upper side of ventricular septum and below right aortic sinus, and severe eccentric AR as well as the isolation of Treatment with vancomycin was initially adopted. However, surgical interventions including removal of vegetation, abscess and occluder, closure of VSD with a pericardial patch, tricuspid valvuloplasty, and aortic valvuloplasty were ultimately performed because of recurrent fever and a new-onset complete atrioventricular block 12-days later. The child continued with antibiotic therapy up to six weeks post operation.The child's temperature gradually returned to normal with alleviation of AR (mild) and heart block (first degree). The following course was uneventful.Late-onset IE could occur following device closure of PmVSD and be associated with post-procedure AR. For non-significant AR after device closure of PmVSD, early surgical intervention could be an alternative for reducing the aggravation of aortic valve damage and the risk of associated IE. Most of them occurred early and a high proportion is temporary and non-significant. Despite presence of a foreign body, post-procedure infectious endocarditis (IE) is considered an extremely rare complication following device closure of PmVSD. Up to now, only two cases have been reported.,4 All of them occurred early after procedure and are speculated resulting from introduction of microbes during implantation, incomplete device endothelialization or residual shunt.,4 Theoretically, AR could increase risk for post-interventional IE. No instances, however, have been reported. Herein, We firstly described a case of very late-onset IE complicated by AR occurring 11 years following device closure of PmVSD with modified symmetrical double-disk device, underscoring importance and necessity of long-term, perhaps life-long follow-up for these patients, and illustrates need for reassessing long-term prognostic implications of non-significant post-procedure AR after device closure of PmVSD and the most appropriate treatment strategy.Transcatheter closure of perimembranous ventricular septal defect (PmVSD) has been proved to be a safe and effective alternative to surgery in selected patients.2Informed written consent was obtained from the parents after the nature of this study had been fully explained to them. The parents of patient have provided informed consent for publication of the case.3 The device was released without any complications such as residual shunt and AR. Oral administration of aspirin (3–5 mg/kg daily) was initiated for 6 months and the child was subjected to 72 h of dynamic electrocardiogram (ECG) monitoring, as well as a 12-lead ECG and echocardiography at 1, 3, and 7 days post procedure. After discharge, he was followed up clinically as well as with 24-h dynamic ECG, and echocardiography at 1, 3, 6, and 12 months during the first year and annually thereafter. A new-onset mild eccentric AR was noted on transthoracic echocardiography (TTE) examination 1-year post procedure, without progression and left ventricular enlargement until latest follow-up in January 2018  11 years ago in our hospital. The procedure was undertaken under general anesthesia and performed in a standard way detailed in our previous study.Staphylococcus aureus was isolated from all three-blood cultures. Treatment with vancomycin was continued since the isolates were resistant to penicillin but sensitive to vancomycin. The origin of this infection was not found. Results of a dental checkup performed during hospitalization were normal. The patient ultimately underwent an emergent surgical intervention because of recurrent fever and a new-onset complete atrioventricular block 12-days later. During the procedure, contracture of right coronary valve, fusion of upper margin of occluder with right coronary valve, perforation of interventricular septal abscess below right coronary valve and vegetation on TV were documented. Removal of vegetation, abscess and occluder, closure of VSD with a pericardial patch, tricuspid valvuloplasty and aortic valvuloplasty were performed. The child continued with antibiotic therapy up to six weeks post operation. The temperature gradually returned to normal with alleviation of AR (mild) and heart block (first degree). The following course was uneventful.On admission, the child was conscious with temperature of 39°C, heart rate of 78 per minute, respiratory rate was 22 per minute, and blood pressure was 114/53 mm Hg. Physical examination was only remarkable for a diastolic murmur in auscultation area of aortic valve. Laboratory tests revealed an elevated white blood cell count of 15.89 × 109/L, neutrophil percentage of 80.1%, platelet count of 527 × 109/L, C-reactive protein of 36.3 mg/L, erythrocyte sedimentation rate of 62.0 mm/h, and mild anemia (hemoglobin: 116 g/L). Transesophageal echocardiography (TEE) demonstrated a vegetation (14 × 4 mm) attached to tricuspid valve (TV), an anechoic area (8 × 7 mm) on left upper side of ventricular septum and below right aortic sinus, and severe eccentric AR. The diagnosis of IE was established. Three sets of blood cultures were taken and empiric antibiotic therapy consisting of vancomycin and ceftriaxone was initiated. 4 device-associated endocarditis following PmVSD closure is scarce. Up to now, only two cases with post procedure IE occurred 10 days and 4 months after transcatheter closure of PmVSD with Nit-Occlud Le device, respectively, have been reported. Both of them were not associated with post-procedure AR. To the best of our knowledge, this is the first case of IE complicated by AR following percutaneous PmVSD occlusion with modified symmetric double-disk occluder. A recent meta-analysis reported 106 patients across 34 studies presented with AR after device closure of PmVSD, with a pooled incidence of 2.0%. Given the worldwide acceptance and increasing utilization of device closure for PmVSD, there are likely additional patients with delayed incorporation that could present a late risk for AR and IE. This study was of clinically amount significance for illustrating the requirement of re-estimating the prognostic implication of non-significant AR after PmVSD device closure and taking the most reasonable deals into consideration for this complication.Even though native VSD endocarditis is well known,,8 few followed the current guidelines and regular follow-up were chosen for these patients by most clinicians since the risk of antibiotic-associated adverse events may outweigh the benefits and most importantly, IE associated with AR after device closure of PmVSD has never been reported before.Actually, apart from severe arrhythmias including high degree or complete atrioventricular block and complete left bundle branch block, post-procedure AR is always another major issue of concern following device closure of PmVSD due to the possibilities of occluder impingement on aortic valve. However, most of the AR was clinically non-significant and may not lead to heart enlargement. For the non-significant AR following device closure of PmVSD, although lifelong antibiotic prophylaxis has been recommended for any type of congenital heart defect repaired with a prosthetic material, whether placed surgically or by percutaneous techniques if valvular regurgitation remains,Our case suggested that early surgical intervention could be alternatively considered even for non-significant AR and it may be a better and more satisfactory means for reducing the aggravation of aortic valve damage and the risk of associated IE.In conclusion, the development of AR resulted from impingement of occluder on aortic valve and associated late-onset IE after device closure of PmVSD in this patient are both causes for concern. For non-significant AR after device closure of PmVSD, early surgical intervention may be a better alternative compared with lifelong antibiotic prophylaxis or no interventions, since associated IE can indeed occur despite its rarity and the risk of antibiotic-associated adverse events may outweigh the benefits.Resources: Kaiyu Zhou, Yimin Hua, Chuan Wang.Supervision: Kaiyu Zhou, Yimin Hua, Chuan Wang.Writing – original draft: Changqing Tang.Writing – review & editing: Changqing Tang, Kaiyu Zhou, Chuan Wang.


FLT3 internal tandem duplication (ITD) mutations are found in around 25% of all acute myeloid leukaemia (AML) cases and is associated with shorter disease‐free and overall survival. Previous reports have shown that FLT3‐ITD induces a specific phenotype in leukemic blasts, which is characterized by high levels of CD33 and CD123, and that expression of CD33 and CD123 is directly influenced by the DNA FLT3‐ITD/wild‐type FLT3 allelic ratio (AR).FLT3‐ITD and 104 FLT3‐ITD–negative AML patients were analysed. Immunophenotyping data were used to calculate antigen expression levels as the ratio between the geometric mean fluorescence intensities (MFIs) of leukemic blasts and MFIs of negative lymphocyte populations. FLT3‐ITD‐DNA and RNA analysis was performed, under the same conditions, by capillary electrophoresis.A total of 42 FLT3‐ITD cohort presented significantly higher CD7, CD33 and CD123 levels. In order to assess the impact of FLT3‐ITD abundance on antigen expression, the patients were grouped for each parameter into two cohorts using the following threshold values: (a) 0.5 for the AR, according to current AML guidelines; (b) 0.7 for the FLT3‐ITD/FLT3‐WT mRNA ratio (RR); and (c) 1.3 for the FLT3‐ITD RR/AR ratio. We found higher values of CD33 for RR/AR ≥1.3, and no other statistical differences between CD7, CD33 and CD123 levels of the other FLT3‐ITD groups. In terms of correlations between MFI values and FLT3‐ITD parameters, we only observed a moderate interdependence between CD33 MFI and the RR/AR ratio, and a weak negative correlation between CD123 MFI and AR.Compared with the control group, the FLT3‐ITD mutations induce a specific antigen profile in AML blasts, and our data do not onfirm previous reports of FLT3‐ITD AR influencing both CD33 and CD123 expression. Also, FLT3‐ITD AML cells have a characteristic high CD33 and CD123 expression,FLT3‐ITD/FLT3‐WT allelic ratio (AR).At diagnosis, FLT3‐ITD AML from our local patient population, and evaluated the impact of both DNA and mRNA FLT3‐ITD/FLT3‐WT ratios on antigen expression levels.In this study, we investigated the quantitative expression of cell surface markers in 22.1FLT3‐ITD mutation, diagnosed between March 2016 and June 2019, and a control group of 104 FLT3‐ITD–negative AML patients were included in this study. All patients gave written informed consent, and the study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Patient demographic details are presented in Appendix A total of 42 AML patients with the 2.2FLT3‐ITD blast expression profile: CD4, CD7, CD9, CD13, CD14, CD33, CD34, CD56, CD64, CD71, CD117 and CD123. Antigen expression levels were calculated as the geometric mean fluorescence index (MFI) ratios between a blast population and a lymphocyte population negative for the respective marker, as previously describedAll primary diagnostic samples, bone marrow aspirate or peripheral blood samples, were processed and analysed according to EuroFlow recommendations.2.3FLT3‐ITD DNA and mRNA qualitative and quantitative analyses by capillary electrophoresis, as previously described.FLT3‐ITD parameters were calculated: the FLT3‐ITD‐to‐FLT3‐WT (a) allelic ratio (AR), (b) mRNA expression ratio (RR) and the (c) relative abundance of FLT3‐ITD mRNA to DNA calculated as the RR/AR ratio. Details regarding the assay and FLT3‐ITD parameters are presented in Appendix We performed 2.4See details in Appendix 33.1FlT3‐ITD–positive cases presented a significantly higher CD7, CD33 and CD123 expression when compared to the FLT3‐ITD–negative control group  0.5 for FLT3‐ITD AR    Figure ,E. There) Figure . There wFLT3‐ITD RR/AR ratio, we evaluated the repartition of NPM1 mutations within the two RR/AR groups , given that CD33 values are also elevated in NPM1‐mutated AMLs.NPM1 mutant cases (11 positive/15 negative cases) than the low RR/AR (<1.3) group (6 positive/16 negative) .To further investigate the relation between CD33 MFI values and the NPM1 mutations, we evaluated the relationship between RR/AR ratio values and NPM1 status by performing a receiver operating characteristic (ROC) curve .Given that patients with high RR/AR ratios had a higher percentage of e Figure . There w4FLT3‐ITD correlated with a specific antigen profile, characterized by higher expression of CD7, CD33 and CD123 when compared to the FLT3‐ITD–negative control population. Increased CD33 and CD123 expression has been previously reported in FLT3‐ITD leukaemic cells.In our study population, the presence of FLT3‐ITD quantitative parameters of leukaemic cells and antigen expression, we also observed a moderate correlation between CD33 and RR/AR values  when compared to the low RR/AR group (<1.3) Figure . When wes Figure ,E. HowevFLT3‐ITD primary leukaemia cells after exposure to FLT3 tyrosine kinase inhibitors, promoting cell survival but not proliferation. Also, in a previous paper by Choudhary et al,Flt3‐ITD–overexpressing 32D cells, the addition of IL‐3 ligand showed to provide antiapoptotic signalling through STAT5a/b activation. One of the main hallmarks of FLT3‐ITD signalling is the aberrant activation of the STAT5 transcription factor.FLT3‐ITD patient population, cases with a lower AR are more dependent of CD123 signalling, with further studies being required to evaluate this hypothesis.In contrast to a previous report of an association between higher AR values and higher CD33 and CD123 MFI values,FLT3‐ITD mutations were associated with a specific antigen expression profile consisting of high MFI values for CD7, CD33 and CD123. However, antigen expression levels are not obviously influenced by FLT3‐ITD DNA and mRNA quantitative parameters. These results suggest that AML patient populations can be further stratified based on differences in phenotype, as well as subtle differences in the genotype of leukaemic cells.In our patient population, There are no conflicts of interests to declare.DSS and ID performed the research. DSS, ER, VP, HB and AMV designed the research study. ER and HB contributed essential tools. DSS and ER analysed the data. DSS and ER wrote the manuscript.Appendix S1Click here for additional data file.


The noradrenergic system is proposed to play a prominent role in the pathogenesis of liver fibrosis. While α1- and β-adrenergic receptors (ARs) are suggested to be involved in a multitude of profibrogenic actions, little is known about α2-AR-mediated effects and their expression pattern during liver fibrosis and cirrhosis. We explored the expression of α2-AR in two models of experimental liver fibrosis. We further evaluated the capacity of the α2-AR blocker mesedin to deactivate hepatic stellate cells (HSCs) and to increase the permeability of human liver sinusoidal endothelial cells (hLSECs). The mRNA of α2a-, α2b-, and α2c-AR subtypes was uniformly upregulated in carbon tetrachloride-treated mice vs the controls, while in bile duct-ligated mice, only α2b-AR increased in response to liver injury. In murine HSCs, mesedin led to a decrease in α-smooth muscle actin, transforming growth factor-β and α2a-AR expression, which was indicated by RT-qPCR, immunocytochemistry, and Western blot analyses. In a hLSEC line, an increased expression of endothelial nitric oxide synthase was detected along with downregulated transforming growth factor-β. In conclusion, we suggest that the α2-AR blockade alleviates the activation of HSCs and may increase the permeability of liver sinusoids during liver injury. Chronic liver disease leads to the dysregulation of various neuroendocrine systems, including the adrenergic system. A significant systemic increase in norepinephrine (NE) and epinephrine in patients with liver cirrhosis and portal hypertension has been known since the 1980s  induced liver injury through α-adrenergic signaling. This is evidenced by the protective effects of a nonselective α1 and α2 blocker (phentolamine) in CCl4-treated mice, which were reflected by decreased tissue necrosis, lactate dehydrogenase (LDH), liver enzymes, and hepatic lipid peroxidation [The role of α-ARs in healthy and diseased livers has been controversial in the literature. The sympathetic nervous system has been suggested to control oxidative stress during carbon tetrachloride  secretion from Kupffer cells, which is inhibited by the α2-AR inhibitor yohimbine ,7. NE in4- or bile duct ligation (BDL)-induced fibrosis and in culture-activated HSCs with and without the α2 blocker mesedin.In light of the aforementioned controversial discussion around the role of α2-ARs in liver fibrosis/cirrhosis, we sought to investigate the influence of the novel α2-AR blocker mesedin (2-(2-methyl-amino-thiozolyl)-1,4-benzodioxane hydrochloride)  on the t2.Murine HSCs (M1-4HSCs) and a human hepatic sinusoidal endothelial cell (hLSEC) line were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose (4.5 g/L) containing either 5% (for hLSECs) or 10% fetal calf serum (for M1-4HSCs), 1% nonessential amino acids (only for M1-HSCs), 100 U/mL penicillin, and 100 µg/mL streptomycin . Cells were kept at 37 °C in an atmosphere containing 5% CO2 culture flasks at a density of 2 × 106 cells/flask. To culture the hLSECs, flasks precoated with collagen 1  were used. After 24 h, adherent cells were incubated for 48 h with or without 10 µM mesedin . Mesedin has been previously characterized as a selective α2-AR blocker [For Western blot analyses, M1-4HSCs and hLSECs were seeded into 150-cmFor immunofluorescence analyses, M1-4HSCs and hLSECs were placed on coverslips in a petri dish at a density of 100,000 cells/coverslip. For hLSECs, the cover slips were precoated with collagen 1. After 24 h, adherent cells were incubated for 48 h with or without 10 µM mesedin.4 in mineral oil or vehicle twice weekly for 4 weeks, as described previously [All animal experiments were approved by the Local Institutional Committee for Animal Welfare in Aachen . For chronic liver injury tests, we employed 6–8-week-old female C57BL/6J mice , which were subjected to an intraperitoneal injection of 0.8 µL/g body weight CCleviously . The micn = 6 each). A common BDL procedure was performed following standardized protocols [For BDL, eight-week-old male C57BL/6J mice were used. The animals were separated into BDL and sham-operated (SO) groups  as a housekeeping gene) were performed using a 7900 Real-Time PCR System (Applied Biosystems) and predesigned TaqMan gene expression assays . Per sample, 1 µg of total RNA was transcribed to cDNA using a high-capacity cDNA reverse transcription kit with an RNase inhibitor (according to the manufacturer’s instructions) . cDNA measurements of α-smooth muscle actin (α-SMA) and the α2-AR subtypes α2a-, α2b-, and α2c-AR  containing “complete protease inhibitor”  was used. A DC Protein assay  was employed to determine the total protein amount. Proteins were fractionated by SDS/PAGE (12% acrylamide) and transferred onto PVDF (Polyvinylidenfluorid) membranes . Membranes were then blocked in 5% BSA   in TBST  for 1 h and incubated overnight at 4 °C with primary antibodies diluted in 5% BSA . The cells were then covered with Vectashield mounting medium  containing 4′,6 diamidino-2-phenylindole (DAPI) and were dried and stored at −20 °C. As for negative controls, samples were treated with secondary antibodies only.For immunofluorescence analyses, hLSECs and M1-4HSCs were each grown on coverslips and incubated with or without mesedin, as described in ® .Immunofluorescence staining was evaluated by fluorescence microscopy using an Olympus BX51 Microscope . Images were acquired using the digital camera F-View II and processed by the software Analysis DOKUPrimary and secondary antibodies were applied in concentrations in accordance with the manufacturer’s information.t-test with GraphPad Prism Software . A threshold of p < 0.05 was considered significant.Data were analyzed using two-tailed Student’s 4 treatment vs the respective controls  using qPCR in the livers of mice four weeks after BDL or CClcontrols A–F. WhilNext, we assessed the influence of mesedin, mediated by α2 blockade, on the expression of α2 receptors in HSCs in vitro. We used M1-4HSC cells, which displayed key features of the intermediate activation of HSCs, which were reflected by α-smooth muscle actin (α-SMA), pro-collagen I expression, and the capacity to undergo a TGF-β-induced transition into a myofibroblastic cell type . In M1-4At the mRNA level, only the α2a and α2b receptors were significantly decreased in mesedin-treated M1-4HSCs A,B, whilThe decrease in α-receptors was concomitant with the downregulation of α-SMA mRNA A and a rImmunofluorescence and Western blot analyses revealed a decrease in TGF-β/α-SMA-positive HSCs  were increased. In contrast, in acute BDL-caused injury, only α2b was elevated. HSCs responded to mesedin with a decrease in key fibrotic markers such as TGF-β and α-SMA. Notably, a concomitant downregulation in α2a-AR and α1-AR was detected in mesedin-treated HSCs. These data may reflect that α2a-AR and α1-AR are at least partially involved in the profibrogenic action of NE, which is produced and released by HSCs [4 model of fibrosis and patients with cirrhosis [The present study reports for the first time the antifibrotic effects of the novel α2-AR blocker, mesedin, in HSCs and its capacity to increase the permeability marker eNOS in hLSECs. In chronic CCl by HSCs , while βirrhosis . Studiesirrhosis . MesedinThe α2-AR blockade  has been reported to influence the generation and release of profibrogenic factors. The direct activation of α2-AR by NE has led to a dramatic increase in TNFα in Kupffer cells, which was reversed by the α2-AR antagonist yohimbine . Our datS-nitrosoglutathione [PDGF has been shown to desensitize α1-AR through phosphorylation . Mesedintathione , our datThe notion that NE substantially contributes to the progression of liver fibrosis is supported by reports showing elevated levels of NE during nonalcoholic fatty liver disease (NAFLD) or after sepsis or hemorrhagic shock ,31. In aOne of the most important hallmarks of liver fibrosis/cirrhosis is the decrease in sinusoidal endothelial cell permeability . In meseHypoxia has been shown to affect approximately one-third of patients with chronic liver disease and is considered to be a prognosis-worsening factor . In mice


Gestational diabetes mellitus (GDM) is a condition associated with the onset of abnormal glucose tolerance during pregnancy. Long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and genes can form lncRNA-mediated feedforward loops (lnc-FFLs), which are functional network motifs that regulate a wide range of biological processes and diseases. However, lnc-FFL network motifs have not been systematically investigated in GDM, and their role in the disease remains largely unknown. In the present study, a global lnc-FFL network was constructed and analyzed. Glycometabolism- and hormone-related lnc-FFL networks were extracted from the global network. An integrated algorithm was designed to identify dysregulated glycometabolism- and hormone-related lnc-FFLs in GDM. The patterns of dysregulated lnc-FFLs in GDM were complex. Moreover, there were strong associations between dysregulated glycometabolism- and hormone-related lnc-FFLs in GDM. Core modules were extracted from the dysregulated lnc-FFL networks in GDM and showed specific and essential functions. In addition, dysregulated lnc-FFLs could combine with ceRNAs and form more complex modules, which could play novel roles in GDM. Notably, we discovered that the dysregulated lnc-FFLs were enriched in the thyroid hormone signaling pathway. Some drug-repurposing candidates, such as hormonal drugs, could be identified based on lnc-FFLs in GDM. In summary, the present study highlighted the effect of dysregulated glycometabolism- and hormone-related lnc-FFLs in GDM and revealed their potential for the discovery of novel biomarkers and therapeutic targets for GDM. Gestational diabetes mellitus (GDM), or glucose intolerance with first onset and recognition in pregnancy, is a common disease among pregnant women . AlthougMarked changes occur during maternal metabolism, and many of these are essential for healthy fetal growth and development. However, they may also lead to GDM and other diseases if there is a substantial deviation from the physiological gestational levels , 7. PregRecently, genetic studies have focused on long non-coding RNAs (lncRNAs), which are defined as non-coding RNAs more than 200 nucleotides in length . lncRNAsIn the present study, a global lnc-FFL network was constructed and analyzed. Glycometabolism- and hormone-related lnc-FFL networks were extracted, and it was shown that lncRNAs play essential roles in these two networks. An integrated computational approach was designed to identify dysregulated glycometabolism- and hormone-related lnc-FFLs in GDM patients. The patterns of dysregulated glycometabolism- and hormone-related lnc-FFLs in GDM are complex. There are strong associations between dysregulated glycometabolism- and hormone-related lnc-FFLs in GDM. Some core modules were discovered and extracted from dysregulated lnc-FFLs in GDM. These dysregulated lnc-FFLs showed specific and essential functions. Furthermore, the dysregulated lnc-FFLs and ceRNAs could form more complex modules that play additional roles in GDM. In particular, we discovered that these dysregulated lnc-FFLs focused on the thyroid hormone signaling pathway. Some drug repurposing candidates were also identified based on lnc-FFL in GDM. Collectively, the present study highlighted the effect of dysregulated glycometabolism- and hormone-related lnc-FFLs in GDM and revealed their potential as novel biomarkers and treatment targets in GDM.http://www.cytoscape.org/). The analysis of the topological features of the network was also performed in cytoscape.Data for experimentally validated gene–miRNA interactions were obtained from miRTarBase 7.0, which is a public database that contains more than 360,000 miRNA–target interactions . For theAll the GDM and normal glucose-tolerant (NGT) samples were obtained from a previous study . All thewww.ncbi.nlm.nih.gov/geo). Whole blood was collected from these 16 subjects in PAXgene Blood RNA Tubes  after overnight fasting. Next, total RNA was isolated using the PAXgene Blood miRNA kit  according to the manufacturer's specifications. Paired data were adjusted for mid-pregnancy weight gain and pregnancy week (GSE92772). The Illumina HiSeq 2500 sequencer was used to obtain the blood cell RNA-sequencing data from the above 16 samples. Adapter trimming was performed using trim_galore/cutadapt v 0.4.0 with standard settings for Illumina reads. Read mapping against the hg38 genome was performed using STAR aligner v 2.4.1d with standard settings. The expression and detailed information about the dataset can be obtained from the GSE92772 database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE92772).The high-throughput expression profiles of lncRNAs, miRNAs, and genes for GDM and normal control samples were obtained from the Gene Expression Omnibus database  for each gene–lncRNA, gene–miRNA, and miRNA–lncRNA interaction in the GDM and NGT samples. For each interaction, the absolute difference between PCC values from the GDM and NGT samples was defined as the difference level for that specific interaction. Thirdly, for each lnc-FFL, two comprehensive scores (CSs) containing differential expression P-values (CSdif) and PCCs (CSPCC) were defined:We designed a comprehensive computational approach to identify significantly dysregulated lnc-FFLs for GDM using global lnc-FFL network and expression profile data. This integrated approach focused on whole change of the L-FFL rather than a single molecule. Firstly, we performed a Student lncRNA, PmiRNA, and Pgene denote the p-values for the differential lncRNA, miRNA, and gene expression, respectively, for each lnc-FFL. CSdif represents the integrated differential lncRNA, miRNA, and gene expression levels between the GDM and NGT samples. GDMlnc−miR, GDMlnc−gene, and GDMmiR−gene represent the PCC values of lncRNA-miRNA, lncRNA-gene, and gene-miRNA interactions, respectively, in GDM patients. Similarly, NGTlnc−miR, NGTlnc−gene, and NGTmiR−gene, represent the PCC values of lncRNA–miRNA, lncRNA–gene, and gene–miRNA interactions, respectively, in NGT samples. CSPCC represents the difference in the co-expression levels of an lnc-FFL between GDM and NGT. Fourthly, we used the values of CSdif and CSPCC to rank all lnc-FFLs based on an equal weighted ranking method, and each lnc-FFL was then given a final comprehensive score. Finally, we performed 1,000 random permutations of the sample labels in the lncRNA, miRNA, and gene expression profiles. Then, the significant lnc-FFLs were obtained by comparing their final comprehensive score with their permutation score (p < 0.05).where Phttp://apps.cytoscape.org/apps/ClusterONE). ClusterONE is a package that clusters a given network based on its topology in order to identify densely connected regions.Key modules containing the node with the highest degree and its neighbors were extracted. Core modules were extracted from the dysregulated lnc-FFL network associated with GDM using the package ClusterOne in cytoscape with default parameters , which is a major topological feature of biology network (lnc-FFL is defined as a regulatory motif whereby a gene regulates an lncRNA and an miRNA while the miRNA also regulates the lncRNA. Multiple lnc-FFLs could form an lnc-FFL network. In our study, a global lnc-FFL network was constructed based on data describing experimentally verified gene–lncRNA, gene–miRNA, and miRNA–lncRNA interactions . The glo network . This in network , 10. We  network . In orde network . The glydif and CSPCC as well as a final score were used to characterize the networks. All three scores showed a similar distribution in both glycometabolism- or hormone-related lnc-FFL networks (dif and the final score approximated a unimodal distribution, whereas CSPCC followed a bimodal-peaks distribution. Notably, we discovered that the dysregulated patterns of lnc-FFLs are multiple and complex. For example, in the dysregulated glycometabolism-related lnc-FFL SP1/miR-200c-3p/CYP1B1-AS1, the interaction between miR-200c-3p and CYP1B1 changed from a positive correlation to a negative correlation (A comprehensive algorithm was developed to identify significantly dysregulated lnc-FFLs in the GDM-associated glycometabolism- and hormone-related lnc-FFL using the global lnc-FFL network and lncRNA, miRNA, and gene expression profiles. We identified 11 glycometabolism- and 29 hormone-related lnc-FFLs that were dysregulated in GDM . Then, tnetworks . Moreoverelation , whereasrelation , and theIn order to explore the associations between dysregulated glycometabolism- and hormone-related lnc-FFLs, we further analyzed both the common and unique dysregulated glycometabolism- and hormone-related lnc-FFLs in GDM. We identified 10 common glycometabolism- and hormone-related dysregulated lnc-FFLs in GDM . An lnc-In order to further characterize the roles of dysregulated lnc-FFLs in GDM, we analyzed the lnc-FFL modules extracted from common glycometabolism- and hormone-related dysregulated lnc-FFLs. First, we extracted a key module, which contained the SP1 gene exhibiting the highest degree and all its neighbors . This keBased on functional analyses, we found a key pathway, namely, the thyroid hormone signaling pathway, which is related with dysregulated lnc-FFLs in GDM . The thylncRNAs, miRNAs, and coding genes can form many types of motifs that play important roles in many biological processes and a large number of diseases. At present, there are many studies focusing on ceRNAs and FFLs. Wu et al. constructed a comprehensive ceRNA network and identified key molecules in diabetic retinopathy . Ling etGDM is a disease characterized by metabolic dysfunction that manifests during pregnancy. Typically, alterations in lipid, glucose, and carbohydrate metabolism occur in pregnant women with GDM . In the Pregnancy alters normal thyroid function, and severe maternal hypothyroidism has been associated with an increased risk for GDM . The levAnother important network motif that contributes to the development of many diseases involves ceRNA . In our In summary, in the present study, a global lnc-FFL network was constructed. An integrated algorithm was designed and significantly dysregulated glycometabolism- and hormone-related lnc-FFLs were identified in connection with GDM. The patterns of the dysregulated lnc-FFLs associated with GDM are multiple and complex. The identified glycometabolism- and hormone-related lnc-FFLs showed strong association with GDM. Core modules with specific functions in GDM could be extracted from the dysregulated lnc-FFLs networks. In addition, dysregulated lnc-FFLs could form more complex modules through combination with ceRNAs. Drug repurposing candidates, such as hormone-related drugs, were identified based on lnc-FFLs associated with GDM. Our study provides insight for the identification of novel biomarkers and drug candidates for GDM.All data generated or analyzed during this study are included in this published article.NL and XF conceived and designed the experiments. HC, SZ, YL, and TL analyzed the data. NL and YS wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.