Dental caries is a major oral health issue which compromises oral health, as it is the main cause of oral pain and tooth loss. Early caries detection is essential for effective clinical intervention. However, methods commonly employed for its diagnosis often fail to detect early caries lesions, which motivates the research for more effective diagnostic solutions. In this work, the relative permittivity of healthy permanent teeth, in caries-prone areas, was studied between 0.5 and 18 GHz. The reliability of such measurements is an important first step to, ultimately, evaluate the feasibility of a microwave device for caries detection. The open-ended coaxial probe technique was employed. Its performance showed to be compromised by the poor probe-tooth contact. We proposed a method based on applying coupling media to reduce this limitation. A decrease in the measured relative permittivity variability was observed when the space between the probe tip and tooth surface was filled by coupling media instead of air. The influence of the experimental conditions in the measurement result was found to be less than 5%. Measurements conducted in ex vivo teeth showed that the relative permittivity of the dental crown and root ranges between 10.0–11.0 and 8.0–9.5, respectively. Dental caries is a complex disease, characterized by progressive destruction of dental hard tissues due to demineralization of the inorganic component and disintegration of the organic constituents of the tooth by acidic by-products, produced from bacterial fermentation from dietary carbohydrates . This isCaries detection is commonly assessed through subjective visual and tactile examination, often supported by X-rays . These mOver the last decades, microwave sensing and imaging has been investigated as a novel diagnostic technique, as it is a low health-risk method due to the application of nonionizing, low-power electromagnetic signals in the frequency range of hundreds of megahertz to a few gigahertz ,18. MicrAmong currently available methods for dielectric properties measurements of biological tissues, the open-ended coaxial probe (OECP) technique is the most commonly used method due to its simplicity and possibility to perform both ex vivo and in vivo measurements over a broad frequency range . HoweverThe small number of experimental studies carried out in the field of dentistry and their limitations motivate further characterization of human teeth dielectric properties. The knowledge about these properties and the level of dielectric contrast between healthy and carious teeth will allow for the feasible evaluation of microwave-sensing technology for early caries detection. Our study focuses on the measurement of relative permittivity of healthy permanent human teeth with OECP technique. As the performance of the probe may be limited by the quality of contact produced between the probe tip and the tooth surface, OECP suitability for dielectric measurement of teeth was evaluated through a measurement repeatability test. A method based on the application of thin layers of coupling media between the probe tip and the tooth surface was proposed to reduce errors induced by poor probe–sample contact. The proposed methodology was applied to estimate, ex vivo, relative permittivity of four healthy permanent teeth in caries-prone areas.In This section describes the dielectric measurement system and sample characterization. Then, the repeatability study conducted to evaluate OECP performance for teeth dielectric characterization is detailed, and the methodology to estimate the tooth’s relative permittivity from measurements conducted with coupling medium application is presented. We overview the measurements made to evaluate the performance of the method that uses coupling media and the identification of potential confounders such as the irregularity of teeth, the amount of coupling media,uo05+ and measurement time. In addition, the application of the proposed methodology to measure relative permittivity of healthy permanent teeth is explained.11) measured by the VNA were converted to complex permittivity by Keysight Material Measurement Suite software. During the measurements, 101 linearly spaced points were measured between 500 MHz and 18 GHz, the frequency range supported by the VNA, an IF bandwidth of 30 Hz. To facilitate the positioning of the human teeth under the probe surface during measurements, teeth were placed into plasticine holders coated with cling film, as shown in The measurement of relative permittivity was conducted using an OECP  connected to a Vector Network Analyzer (VNA)  through a coaxial cable a. The reThe acquisition system was calibrated by the measurement of reflection coefficients: of an open circuit, i.e., the probe surrounded by air; of a short circuit, produced by connecting a short-block to the probe; and of distilled water. The quality of the calibration and its maintenance through the acquisitions was estimated from the comparison between the measured and modelled relative permittivity of the 0.1 M NaCl solution , according to Equation (1):al model , and ε′BIn our study, healthy permanent teeth without prior restoration or root devitalization were used. All samples were extracted by dentists at Malo Dental as part of patients’ prosthetic treatment, according to the data acquisition protocol established with the clinic. After extraction, the teeth were cleaned with a physiological solution to remove biological fluids and hydrogen peroxide for disinfection. All samples were sealed in sterilized bags, where they remained until measurement. The samples and the measurement sites, characterized in each of the conducted studies, are summarized in All measurements were conducted at room temperature with dried samples. All samples had a thickness and diameter larger than 5 and 7 mm, respectively. Although the sample diameter was below the one recommended by OECP manufacturer (10 mm) , some stTo evaluate OECP performance for teeth dielectric characterization, a repeatability study was conducted over the crown and the root of two teeth. For each sample, measurements were conducted over the flattest and most regular surfaces of the root and crown in order to maximize the contact between the probe and the tooth. In total, 10 independent measurements were performed for each of the chosen measurement positions. By independent measurement, we mean the repetition of the entire measurement process between two acquisitions, i.e., after a measurement was completed, the probe was detached from the measured point, and the tooth was removed. The probe was cleaned and placed in contact with the desired measurement point in order to perform a new acquisition. All measurements were conducted at room temperature: [20.7 ± 0.2] °C. The validation measurements were performed before and after the completion of each set of 10 acquisitions.f, and The percentage variation of the relative permittivity over the repeatability test was calculated according to Equation (1), where Given the irregular tooth surface, producing a proper contact between the probe and tooth is challenging, and as a result, the measurements are contaminated with air properties. To overcome this limitation, alternative approaches for dielectric teeth characterization have been investigated. Since taking measurements over artificially smoothed tooth surfaces, produced either by polishing or by cutting, lead to sample modification, we considered using coupling media to reduce errors induced by poor probe-sample contact.Studies carried out to date regarding the OECP interaction with heterogeneous tissues ,45 showeIf = f, for which the relative permittivity of the MUT and the coupling medium are similar, for the frequency range near this point, it is expected that the relative permittivity measured by the probe when coupling medium is applied over MUT (shaded band) is similar to the coupling medium relative permittivity (red line). Thus, for example in If = f, will allow for more reliable estimation of the relative permittivity of MUT such as the tooth, minimizing the error introduced in the measurement by the presence of air gaps.For example, if the relative permittivity of the coupling medium and the MUT are those described by red and blue curves in A total of twelve coupling media have been produced by combining different proportions of vegetable glycerin (Alifar Glycerin) and distilled water (W5 Distilled Water), namely 100:0 (G[100]), 95:5 (G[95]), 90:10 (G[90]), 85:15 (G[85]), 80:20 (G[80]), 75:25 (G[75]), 70:30 (G[70]), 65:35 (G[65]), 60:30 (G[60]), 55:45 (G[55]), 50:50 (G[50]) and 45:55 (G[45]), in percentage by volume. For example, according to the above notation, G[70] corresponds to a solution formed by 70% volume of glycerin and 30% volume of distilled water. The relative permittivity of produced solutions, for the temperature of [18.4 ± 0.4] °C, are shown in Glycerin is a non-toxic compound  that entCalibration of measurement equipment.Validation of calibration with 0.1M NaCl solution. Overall, two validation measurements are performed: after equipment calibration and after completion of all measurements.3.1Air relative permittivity measurement to ensure that the probe was properly cleaned and calibrated.3.2Tooth relative permittivity measurement without coupling medium application. This step allowed for one to obtain a reference of the tooth relative permittivity and to check if the tooth was properly clean, and if it had already been placed in contact with the coupling medium in previous measurements.3.3Tooth relative permittivity measurement with coupling medium application. To this end, the probe is detached from the tooth measurement site, a drop of coupling medium is applied to the tip of the probe, and the dielectric properties are measured . The dro3.4The probe and the tooth are cleaned with distilled water.Tooth relative permittivity measurement with application of the coupling medium on the tooth surface. For each combination “coupling medium + tooth” nine measurements were performed according to the following protocol:To determine the dielectric properties of the coupling medium and monitor its changes, its properties are measured at the beginning, middle and end of the nine acquisitions.To estimate teeth relative permittivity by applying coupling media, for each “coupling medium + tooth” combination, the following protocol is applied:Twenty-three acquisitions were performed for each “coupling medium + tooth” combination: two validation measurements; three measurements of the coupling medium properties; nine measurements of the tooth with the coupling medium; and nine measurements of the tooth without the coupling medium. The measurements of each combination were completed, on average, in two hours. Twelve combinations of coupling medium and tooth were performed for tooth crown dielectric characterization and ten combinations for dental root characterization.To estimate the relative permittivity of the teeth, the relative permittivity measured in each of the nine acquisitions for each combination of “coupling medium + tooth” was compared to the average relative permittivity of the tested coupling media. For each acquisition, the intersection point was defined as the frequency for which relative permittivity of the “coupling medium + tooth” was closest to the average relative permittivity of the applied coupling medium. The median value of the relative permittivity recorded at the intersection points was used as an estimation of the relative permittivity of the tooth for the frequency. The application of multiple coupling media with different properties allowed for the identification of several intersection points crosses,  between The measurements conducted to evaluate the performance of the proposed method and to identify the potential confounders are detailed in this subsection. Firstly, the methodology applied to test the influence of the irregularity of the tooth surface, and consequently the quality of probe-tooth contact, on the estimated relative permittivity, is presented. Then, we describe the tests conducted to evaluate the influence of some experimental conditions, such as the amount of the coupling medium applied over the tooth surface or measured relative permittivity variation with time elapsed between the application of the coupling medium and the start of the measurement.To evaluate the dependence of the tooth’s relative permittivity estimation with the quality of OECP contact and, consequently the amount of coupling medium between the probe and the tooth surface, we compared: the estimated relative permittivity over a smooth and flat tooth surface , was calculated between the median measurement, ε′M(f), and the remaining eight measurements, ε′i(f), taken for each “coupling medium + tooth” combination, according to Equation (2):Since the application of equal coupling media for each measurement site (R1 and R2) produced intersections at different frequencies, to quantify the change in relative permittivity recorded between the two measurement sites, the permittivity estimated over R1 was interpolated to the intersection frequencies of R2 for comparison. Thus, the average percentage change of estimated relative permittivity between R2 and R1 sites was computed by averaging the percentage changes obtained for each of the intersection frequencies from the R2 measurement sites. For comparison, percentage variation of the relative permittivity recorded without the application of coupling media  was calculated according to Equation (1), where To study the dependence of the measured relative permittivity given the amount of coupling medium applied to the tooth surface (Test A), we compared: the relative permittivity recorded with the application of the largest amount of the coupling medium feasible to be placed on the tooth surface (a drop attached to the probe tip plus coupling media covering the tooth surface and probe surroundings); the relative permittivity measured by applying the smallest amount of coupling medium (a drop attached to the probe tip), so that it remained only between the probe and the tooth surface. Care was taken not to touch the probe, so that its position was stable. The entire measurement process lasted less than one minute.To study the variation of the measured relative permittivity with the exposure of the coupling medium to the environment conditions, for instance, temperature changes (Test B), we evaluated the variation of measured relative permittivity with time elapsed between the application of the coupling medium and the start of the measurement. For this purpose, we compared: the relative permittivity measured immediately after the application of the coupling medium on the tooth (reference measurement); the relative permittivity measured one minute and twenty seconds after the reference measurement (delayed measurement). This interval was chosen because it is much longer than the time usually taken between the application of the coupling medium on the tooth and the beginning of the measurement (20/30 s).Both tests were conducted with six coupling media with different proportions of glycerine and water . For each combination of “coupling medium + tooth”, the measurement process was repeated three times. All measurements were conducted at room temperature, which was [19.4 ± 0.2] °C for Test A and [19.3 ± 0.5] °C for Test B. The average difference observed between the coupling medium and room temperature at the measurement time was [0.3 ± 0.1] °C. The validation measurements were performed before and after measuring each “coupling medium + tooth” combination.f, respectively, while for Test B, To estimate the error induced by each of the evaluated conditions, the percentage variation of the relative permittivity was calculated according to Equation (1). In Test A, The proposed method was applied for the characterization of four healthy teeth, two premolars (PM1 and PM2) and two molars (M1 and M2) at caries-prone sites: one on the crown (C1), between the middle and cervical thirds of the smooth crown surface; and the other on the cervical third of the tooth root (R1). The locations of the measurement points, for each sample, are illustrated in The selected measurement points were on surfaces as flat and smooth as possible, in order to maximize the probe–tooth contact. In the root, the choice of the cervical third for acquisitions was due to the fact that root caries typically develop on the cervical area, due to exposure of the cementum, after gingival recession ,49. In tThe measurements were conducted according to the procedure described in In order to compare the relative permittivity estimated in the present study with the relative permittivity available in the literature, the relative permittivity percentage change was determined according to Equation (1), where terature , respectIn this section, firstly, the results concerning the OECP performance for teeth dielectric characterization are discussed. Then, the performance of the coupling medium method is presented, and the influence of potential confounders is discussed. Finally, the estimated relative permittivity of healthy permanent teeth is presented.The range of relative permittivity recorded during the repeatability study where the teeth are measured with no coupling media can be found in For the considered frequency range, the recorded relative permittivity average percentage variation ranged from 18.1% to 34.5%, for the considered measurement sites. Furthermore, the highest relative permittivity recorded, for both crown and root, was below the values reported by Hoshi et al.  for the This result highlights the difficulty associated with characterizing teeth dielectric properties with OECP. Thus, if the acquisition system is formed only by a probe in direct contact with the tooth sample, the result will be compromised by the presence of air.According to the proposed methodology, the use of the relative permittivity observed at the intersection points between the relative permittivity measured with the application of the coupling medium on the tooth and the relative permittivity of the medium, as an estimation of the relative permittivity of the tooth, should reduce the error induced by poor contact between the probe and tooth surfaces. Therefore, the frequency range near the intersection point should lead to a reduction of the variability observed between different acquisitions of tooth relative permittivity. These zones would correspond to frequencies where the dielectric properties of the coupling medium and the tooth are similar.The result in The average percentage variation of relative permittivity was estimated with: the amount of coupling medium applied on the tooth surface (Test A); and the time elapsed (t = 80 s) between the application of the coupling medium to the tooth and the start of the measurement (Test B). The variation in the measured relative permittivity, caused by either the amount of coupling medium applied over the tooth surface or by the time elapsed between the application of the coupling medium and the beginning of the measurement, was less than 5%, the highest acceptable error established for dielectric properties measurements. Therefore, it is expected that the conditions under which the measurements were taken, in this study, did not significantly interfere with the reported results.An overlap can be observed between relative permittivity estimated within each of the measurement sets, root (R1) and crown (C1). In addition, a clear distinction is observed between the relative permittivity estimated for the root and the crown, at higher frequencies, which becomes less noticeable for lower frequencies. This pattern is also found in the presence of air a. RegardThis study focused on the measurement of the relative permittivity of healthy permanent human teeth with the OECP technique, whose knowledge is essential to evaluate the feasibility of a caries detection device working in the microwave frequency range. OECP performance in the dielectric measurement of teeth was evaluated, and a method based on the application of coupling media between the probe tip and tooth surface was proposed in order to reduce errors induced by poor probe–sample contact. The experimental protocol was developed, and potential confounders were identified and evaluated. The error introduced by experimental conditions, such as the amount of coupling medium applied over the tooth surface or relative permittivity variation with time elapsed between the application of the coupling medium and the start of the measurement, was found to be comparable to the error introduced by the measurement system, i.e., error below 5%. The quality of contact produced between the probe tip and the tooth surface remained a factor of variability. However, the experimental results showed that the proposed methodology led to a reduction in the variability of measured relative permittivity when the space between the probe and the tooth surface was filled by the coupling medium instead of by air. This outcome highlighted the ability of the developed method to minimize the error associated with poor probe–tooth contact and, therefore, showed the advantage of its employment in the measurement of dielectric properties of irregular solids.The proposed methodology was applied to characterize the relative permittivity of four healthy permanent teeth, ex vivo and between 0.5 and 18 GHz. Measurements carried out at caries-prone areas showed that the relative permittivity of the dental crown varies between 10.0 and 11.0, while the relative permittivity of dental root is found to be between 9.5 and 8.0, for the considered frequency range. No measurements were performed for teeth with caries, limiting the prediction of the usefulness of this method for caries assessment.Future work should comprise increasing the sample size and including teeth with different degrees of caries, in order to assess whether there is a dielectric contrast between healthy and carious teeth, and how this contrast evolves with the extent of tooth decay. Carious teeth may present a large variability of characteristics and, consequently, will have different dielectric properties, mainly due to the caries size, its position and depth within each tooth. In addition, deterioration of the teeth may hamper probe–teeth contact, but this type of caries would be visually detected, and an imaging diagnostic tool would not be required. Since the dielectric properties of tissues vary with temperature and degree of hydration, measurements under conditions closer to those found in the oral cavity should be performed.


Mutations in genes encoding molecular chaperones, for instance the genes encoding the subunits of the chaperonin CCT , are associated with rare neurodegenerative disorders. Using a classical molecular dynamics approach, we investigated the occurrence of conformational changes and differences in physicochemical properties of the CCT5 mutations His147Arg and Leu224Val associated with a sensory and a motor distal neuropathy, respectively. The apical domain of both variants was substantially but differently affected by the mutations, although these were in other domains. The distribution of hydrogen bonds and electrostatic potentials on the surface of the mutant subunits differed from the wild-type molecule. Structural and dynamic analyses, together with our previous experimental data, suggest that genetic mutations may cause different changes in the protein-binding capacity of CCT5 variants, presumably within both hetero- and/or homo-oligomeric complexes. Further investigations are necessary to elucidate the molecular pathogenic pathways of the two variants that produce the two distinct phenotypes. The data and clinical observations by us and others indicate that CCT chaperonopathies are more frequent than currently believed and should be investigated in patients with neuropathies. The chaperone system (CS) of an organism consists of all its molecular chaperones, co-chaperones, chaperone co-factors, and chaperone interactors and receptors . The canOf note is the importance of the CCT5 subunits, not only in the constitution of the whole CCT complex, but also their existence and function as free monomers and as homo-oligomeric complexes ,20,21. DN-terminal (amino acids 1–154) and C-terminal (amino acids 418–541) segments that form loop-A and loop-B for the interaction with the α- and β-phosphate groups of ADP and a sensor loop, also called a stem-loop, located between the β1 and β2 sheets, for the contacts with the substrate    25]. Th. ThN-tertubulin) . The Histubulin) .N-terminal (amino acids 155–226) and the C-terminal (amino acids 381–417) segments, including the α-helixes of the lower hinge, helix-6 and helix-12, which establish contact with the adjacent segments of the equatorial domain, whereas helix-7 contacts the upper hinge. The upper hinge consists of β-sheets, β3, β4, β12  of CCT5 variants and wild type under the three conditions: nucleotide-free, ATP-bound, and ADP-bound. The amino acids of the apical and intermediate domains were the most fluctuating. Notably, the apical and intermediate domains of the Leu224Val variant fluctuated mostly in the absence of nucleotides and in the presence of ADP A,C, wherAlteration of the hydrogen-bond network of molecular chaperones may lead to changes in domain communication, impair peptide binding, and alter ATP hydrolysis capacity. Therefore, we analyzed the predicted number of hydrogen bonds in CCT5 mutants. The two variants showed different amounts of hydrogen bonds in all three conditions, and both differed from the wild type .The His147Arg variant showed the highest number of hydrogen bonds in all conditions. The number of hydrogen bonds in the wild type was highest in the ADP-bound conformation , row 1. Adaptive Poisson–Boltzmann Solver (APBS) software was used to reveal the electrostatic potential of the ATP-bound wild-type CCT5 and the two mutants His147Arg and Leu224Val. The wild-type molecule showed a homogeneous distribution of charges on its surface A; areas The data reported here show that two different missense mutations, His147Arg and Leu224Val, occurring in the equatorial and intermediate domains of CCT5, respectively, can alter the molecular anatomy and physicochemical properties of the subunit. Molecular dynamic simulations revealed the bending of the apical domain toward the equatorial domain of the His147Arg mutant, particularly under nucleotide-free and ATP-bound conditions. On the other hand, the Leu224Val mutation, occurring in the intermediate domain, in the absence of nucleotide, stretches the apical domain, compared to the wild-type subunit, moving the apical domain of the mutant molecule away from its equatorial domain. In the presence of ATP, the apical domain of the Leu224Val mutant returns closer to its equatorial domain than the apical domain of the wild-type subunit, but not as close as the apical domain of the CCT5 His147Arg variant. In the presence of ADP, the molecular models of both variants are similar but differ from that of the wild type. The models show a marked conformational change of the apical domain in both variants, particularly under ATP-bound and nucleotide-free conditions.The RMSF of both CCT5 mutant subunits showed flexibility/fluctuation of the subunit domains, in agreement with data in the literature . HoweverThe apical domains are the most heterogeneous regions of the CCT subunits and have the role of recognizing, interacting, and capturing the substrates . The apiThe different molecular conformations observed between the two variants and the wild-type CCT5 suggest an unusual and instable “starting receptive structure” of the mutants and an abnormal displacements/dynamism of the variants’ domains, especially of the apical domain.The Leu224Val mutation occurs in the β4-sheet at a point that may be considered the intersection between the intermediate and apical domains. The intermediate domain works as a molecular hinge that bidirectionally transmits and integrates the various conformational changes at the apical and the equatorial domains . To dateWe detected other interesting differences between the variants and the wild-type CCT5. Leu and Val are two non-polar hydrophobic amino acids with similar molecular weight, 131.175 g/mol and 117.148 g/mol, respectively. On the other hand, His and Arg are two polar amino acids with a molecular weight of 155.157 g/mol for His, which can, because of its aromatic ring, switch from a positively charged to neutral form, and 174.204 g/mol for Arg, which is always positively charged. Despite the similarities between the substituted amino acids, Leu with Val, and His with Arg, we detected diverse amounts of intramolecular hydrogen bonds in the CCT5 variants , suggestThe charge surface of proteins can act as a direct signal to the proteasome and induce the formation of a specific hydrated surface around the protein . Water mThe structural properties of the monomeric CCT5 subunits, wild type, and mutant, were extracted from the structure of the crystallized homo-oligomeric complex deposited in the Protein Data Bank with accession code 5UYZ .https://server.poissonboltzmann.org/) was used for the calculation of the potential around our molecule (by solving equations of continuum electrostatics) [The structure of the mutant subunit was obtained by changing amino acid 224 leucine with valine, using the software UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311 (accessed on 18 December 2020), . Moleculstatics) . Selectestatics)  and CHIMMany rare genetic, including monogenic, disorders are characterized by a heterogeneity of signs and symptoms differing between patients, which makes diagnosis and pinpointing of the etiopathogenic factor difficult. Consequently, patient management and prognostication are extremely challenging. Efforts toward a better understanding of the molecular mechanisms underlying these pathologies are a promising way to make progress in the management of these diseases. Furthermore, since molecular chaperones are expressed by all the cells of the human body, genetic chaperonopathies are expected to occur with a frequency higher than currently believed, but one must be aware that they may be hidden from routine clinical examination. To remedy this weakness of practical medicine, for the benefit of patients and science, chaperonopathies must be included in medical studies curricula.The data reported here show that two different missense mutations, His147Arg and Leu224Val, occurring in the equatorial and intermediate domains of CCT5, respectively, are predicted to alter the molecular anatomy and physicochemical properties of the subunit in different ways. The replacement of one amino acid with another, even if both are very similar, is predicted to cause disturbances in nearby charges and bonds that, in turn, seem to propagate to distant sites of the CCT5 molecule, e.g., from a molecular domain  to another . These are significant predictions, if we consider that: (1) the apical domain has structural, functional, and regulatory roles; (2) the CCT5 subunit is the starting point for the assemblage of the homo- and hetero-oligomeric complexes; and (3) geometrically specific binding conformations are critical to trigger mechanical inputs to initiate the folding cycle. The pathological result can be predicted to be a defective chaperonin unable to fold substrates, which is most likely the pathogenic mechanism underpinning many of the abnormalities observed in patients’ muscles and nerves.The predictions emerging from the present study should be a valuable guide to the continuation of in vitro and in vivo studies, expanding those reported previously ,24,25,26


Melanoma differentiation-associated gene 9 (MDA-9) is a small adaptor protein with tandem PDZ domains that promotes tumor progression and metastasis in various human cancers. However, it is difficult to develop drug-like small molecules with high affinity due to the narrow groove of the PDZ domains of MDA-9. Herein, we identified four novel hits targeting the PDZ1 and PDZ2 domains of MDA-9, namely PI1A, PI1B, PI2A, and PI2B, using a protein-observed nuclear magnetic resonance (NMR) fragment screening method. We also solved the crystal structure of the MDA-9 PDZ1 domain in complex with PI1B and characterized the binding poses of PDZ1-PI1A and PDZ2-PI2A, guided by transferred paramagnetic relaxation enhancement. The protein–ligand interaction modes were then cross-validated by the mutagenesis of the MDA-9 PDZ domains. Competitive fluorescence polarization experiments demonstrated that PI1A and PI2A blocked the binding of natural substrates to the PDZ1 and PDZ2 domains, respectively. Furthermore, these inhibitors exhibited low cellular toxicity, but suppressed the migration of MDA-MB-231 breast carcinoma cells, which recapitulated the phenotype of MDA-9 knockdown. Our work has paved the way for the development of potent inhibitors using structure-guided fragment ligation in the future. Human proteomes contain approximately 600,000 protein–protein interactions (PPIs)  to mediaThrough the interactions between the PDZ domains and the substrates, MDA-9 tandem PDZ domains are involved in syndecan-mediated signaling to the cytoskeleton and perform a variety of physiological and cellular functions ,16,17, eThe discovery of inhibitors targeting the PDZ domain remains challenging because of the shallow and dynamic binding interface. Despite the discovery of inhibitory peptides or peptidomimetic compounds against PDZ domains, small molecules are highly desirable due to their membrane permeability and reduced risks of immune reactions or biodegradation. PDZ1i, developed by Fisher et al., was demonstrated to be able to bind to the MDA-9 PDZ1 domain with an affinity of 21 μM, but did not bind significantly to the MDA-9 PDZ2 domain. PDZ1i is a suitable potential pharmacological tool for investigating the role of the PDZ1 domain of MDA-9 in cellular mechanisms and vivo efficacy studies. For example, PDZ1i inhibited the invasion of glioma cells and improved survival rate in brain-tumor-bearing mice . PDZ1i a15N-labeled MDA-9 PDZ1 or PDZ2 domain at a ligand/protein molar ratio of 4:1, respectively  restraints. Transferred PRE describes the difference of transverse relaxation rates of the ligand in the presence of the diamagnetically or paramagnetically labeled protein, which was proportional to the inverse sixth power of the distance between the paramagnetic center and the ligand atom of interest . Since t1H spectrum of PI2B had only one proton  revealed three key interactions with conserved residues, V209, G210, and F211, of MDA-9 t and [L]t represent the concentrations of the protein and ligand, respectively. Δδobs represents the observed chemical shift changes, relative to the free-state protein.According to this equation, the binding constants can be best fitted with a binding mode of 1:1:http://www.pymol.org/ accessed on 10 October 2022).The MDA-9 PDZ1 domain was concentrated to 15 mg/mL in a HEPES buffer , then centrifuged at 13,000 rpm for 30 min at 4 °C. We discarded the precipitate. Crystals of the PDZ1 domain, in complex with PI1B, were grown at 4 °C using the hanging drop vapor diffusion by mixing 1 μL of mix with 1 μL of reservoir buffer  chloride hexahydrate, pH 6.5) for 7–15 days. These crystals were flash frozen in liquid nitrogen using a cryoprotectant (20% glycerol added to the reservoir solution). Datasets for X-ray diffraction were collected at the Shanghai Synchrotron Radiation Facility via the BL18U beamline. HKL2000 was used to index, integrate, and scale all datasets. Phenix’s eLBOW software was used to prepare PI1B. As a search model with molecular substitution, Phaser MR4 was used to solve the composite structures of MDA-9 PDZ1 and PI1B. Coot5 was used to model the structure, followed by Refmac56 and Phenix7 for refinement. The crystal diffraction data and refinement statistics are summarized in AutoDock is a powerful tool for molecular docking . Mutant 15N-labeled PDZ1 C118/C166S mutant and the PDZ2 C239S/D256C mutant were expressed by the method described above. After the dilution of the protein solution to 0.5 mg/mL with a 1-fold excess of Tris(2-carboxyethyl) phosphine (TCEP), the solution was incubated for 2 h at room temperature. The protein solution was then incubated overnight at 4 °C with an excess of 5-folds methionine spin label (MTSL). The excess MTSL molecules were removed by dialysis. In order to confirm the chemical attachment of MTSL to the mutant, 1H-15N HSQC spectra were obtained. The MTSL-labeled PDZ1 or PDZ2 domains of the mutants were used to determine that the NMR titration experiments of PI1A or PI2A did not alter the binding mode. MTSL-labeled PDZ1 or PDZ2 mutant proteins (200 μM) were titrated into a solution of PI1A or PI2A (0.2 mM), respectively. WATERGATE spectra were collected using T2 delays of 0.008 and 0.08 s. Vitamin C was then added, in excess of five times the amount of MTSL, and the reaction was incubated overnight. WATERGATE spectra with T2 delays of 0.04 and 0.8 s were recorded. The experimental error was estimated by repeating the experiment. ACD Lab was used to integrate the peaks between 6 and 8 ppm of the WATERGATE spectrum of PI1A or PI2A.The R2 was best fitted to an exponential decay equation as follows:tI and I0 represent the intensities measured at the relaxation T2 delays of t and 0 s, respectively.In this case, Γ2 would be as follows:It was determined that the transferred PRE Rdia)2( and Rpara)2 or the residue D256 (PDZ2) to ith atom of PI1A or PI2A. The constant A was determined by nuclear and protein properties, as well as the bound fractions of PI1A and PI2A.Q value was calculated to assess the agreement between the experimental and calculated Γ2 values:Γ2 values for the 1st atom in PI1A or PI2A, respectively.The y represents the value of anisotropy, x represents the concentration of protein, L represents the concentration of the FITC-peptide, and dK represents the equilibrium dissociation constant of the FITC-peptide.The wild-type or mutant proteins of the MDA-9 PDZ1 and PDZ2 domains were prepared at various concentrations, ranging from 0 to 600 μM, as described above. The FITC-labelled peptide (sequence TNEFYFGSGS) was diluted to 500 nM in a HEPES buffer . The FITC-labelled peptide was mixed with different concentrations of protein and incubated for 30 min. At 25 °C, the samples were excited at a wavelength of 485 nm and detected at a wavelength of 525 nm using a CLARIOstar  plate reader. The experiments were performed in triplicate. The following equation was used to fit the binding constant in a 1:1 binding mode:dK of the compounds for competitive FP was fitted using the following equation [y represents the value of anisotropy, x represents the concentration of the compounds PI1A and PI2A, L represents the concentration of the FITC-peptide, R represents the concentration of protein, K1 denotes the equilibrium dissociation constant of the FITC-peptide, and K2 represents the dissociation constant and indicates the probability that the small molecule to be fitted will compete for binding to the protein with the FITC-peptide.For competitive FP experiments with the FITC-peptide, the PDZ1 domain concentration was set at 80 μM and the PDZ2 domain concentration at 250 μM. After that, each aliquot of the 200 μL sample contained 500 nM of the FITC-peptide, 80 μM of the PDZ1 or 250 μM of the PDZ2 domain, as well as different concentrations of PI1A or PI2A. A concentration range of 0–200 μM was administered for PI1A while a concentration range of 0–600 μM was administered for PI2A. Samples were repeated 3 times for each experimental group. The dissociation constants for the interactions between ligands and proteins were derived from the complete competitive FP equation proposed by Gerhard Wagner et al. (Biochemistry 2004). The equation :(8)y=2× plate reader.The MDA-MB-231 cells were obtained by a gift. CCK8 assay was used to assess the effects of PI1A or PI2A on the viability of MDA-MB-231 cells. Cells were placed in 96-well plates and treated with different concentrations of DMSO, PI1A, or PI2A for 48 h. Then, 10 μL of CCK8 solution was added to the wells. The 96-well plates were incubated in a COMDA-MB-231 cells were spread in 6-well plate culture dishes, where sh-MDA-9 cells were used as a positive control. DMSO (control), PI1A 100 μM, PI2A 100 μM, and both PI1A and PI2A 100 μM were added to the MDA-MB-231 cells, respectively. On newly grown monolayer cells, 10 L of the tip was used to line the central area of cell growth. Next, excess cells were washed away with PBS buffer. The cells were then cultured for the experimentally-determined period of time (0 h or 24 h). Lastly, the cell culture plates were removed separately and the peripheral cells were observed under a microscope to determine whether they had migrated to the scratched area.MDA-9 shRNA and control shRNA were purchased from Open Biosystems. HEK293T cells were seeded in a 6-well dish and then transfected with 1μg of retroviral DNA encoding MDA-9 vector or control vector, 1 μg packaging plasmid mix (0.8 μg psRAX2 and 0.2 μg pMD2.G) to generate lentiviruses. Virus infection was performed in MDA-MB-231 cells for 48 h. Add puromycin to screen for virus-infected cells. Cells were then collected and total RNA was extracted. RNA extraction was performed using SV Total RNA . cDNA was reserved from total RNA by using the First Strand cDNA Synthesis Kit . The Real-time PCR  and SYBR-Green PCR Master Mix  were used for amplification and detection. GAPDH was used as the normalization control of the amplifications.MDA-9 is an adaptive scaffolding protein that exerts its diverse functions by interacting with downstream proteins. MDA-9 plays a crucial regulatory role in various tumorigenic pathways, making it an emerging therapeutic target. Although PDZ1i has been shown to be an effective pharmacological tool to study the biological function of the MDA-9 PDZ1 domain in cells and in vivo, structural studies of its interaction with the MDA-9 PDZ1 domain are absent. In addition, C58 is a weak inhibitor that only targets the MDA-9 PDZ2 domain. The highlight of our study was the screening of inhibitors against the PDZ1 and PDZ2 domains of MDA-9, respectively. Meanwhile, we investigated the interaction pattern of the inhibitors with the proteins using biophysical approaches. The details of these interactions will provide a fine-grained structural basis for the subsequent development of the bivalent state inhibitors of the MDA-9 PDZ tandem. The fragment-screened hit PI1A and PI2A were shown to inhibit the binding of the MDA-9 PDZ domain to natural substrates through competitive FP assays, respectively. Furthermore, both compounds, PI1A and PI2A, demonstrated low toxicity to the MDA-MB-231 cells and impaired cell migration, consistent with the results of MDA-9 knockdown. Overall, we identified two compounds that bound to the natural substrate pocket of the tandem PDZ domains of MDA-9. Using structure-guided fragment linking, we envision the development of potent bivalent state inhibitors that will provide a powerful strategy for cancer therapy in the future.


Crystal integrity is preserved and the deformation is associated with a large bending angle (65.44°). This is a new strategy to increase the magnitude of the dissipated energy in an organic solid comparable to that seen in alloys. By X-ray crystallographic analysis it was determined that a large molecular rearrangement at the twinning interface preserves the crystal integrity. Drastic molecular rearrangement at the twinning interface helps to preserve hydrogen bonding in the molecular rotation, which facilitates the large bending angle. The maximum shear strain of 218.81% and dissipated energy density of 1 MJ m We demonstrate exceptional twinning deformation in a molecular crystal upon application of mechanical stress. This could be due to the typical fragility and tiny size of organic crystals. The dissipated energy (Ed) derived from twinning deformation of alloys and organic single crystals depends on the following factors: (i) coercive stress (σc) and (ii) bending angle (θ), given as (Ed = σc tan θ). In alloys the required magnitude of σc is larger than in the case of molecular crystals, deriving a significant magnitude of Ed. However, the value of θ is usually smaller in alloys than in organic crystals. The Ed value in alloys  is much higher than in typical organic solids (approximately 102 kJ m−3). Application of a relatively smaller σc in the deformation of organic single crystals can produce a large θ without crystal cleavage. Achievement of a large θ paves the way for new design principles to increase the value of Ed in organic single crystals. Moreover, alloys have polycrystalline properties so on average θ is reduced due to their isotropic nature, whereas, in organic single crystals the composition is almost anisotropic and associated with comparatively higher values of θ which is advantageous for achieving enhancing Ed.Twinning deformation is a type of plastic deformation that has been extensively investigated in metal alloys, mostly from the perspective of physics and materials science.2 and tetramethyle-tetraselenafulvalene (TMTSF)2X3 , while among organic solids the following examples have been reported: 1,3,5-tribromo-2,4,6-triiodobenzene,4 1,3,5-trichloro-2,4,6-triiodobenzene,4l-lysine monohydrochloride dihydrate,5 and adipic acid doped with 3-methyl adipic acid.6 Studies of twinning deformation in such organic or organometallic solids are underdeveloped in contrast to the studies in metallic solids7 involving a control of their mechanical durability or deformability.Some of the reports of twinning deformation in single crystals relate to organometallic compounds such as ferrocene8–11 supported with experimental evidence by microscopic observations, X-ray crystal structure analysis, and force measurements. For example, the organosuperelastic crystals of the planar molecule 3,5-difluorobenzoic acid8 showed exceptional twinning. Crystal of 5-chloro-2-nitroaniline9 exhibited twinning ferroelasticity. The flexible rod shaped adipic acid molecule showed ferroelasticity driven by conformational change.10 Finally, a non-planar 4,4′-dicarboxydiphenyl ether11 exhibited ferroelasticity by partial ring flipping. However, the bending angles in these crystals were 27.8°, 49.21°, 44.65°, and 16.9°, respectively. Moreover, the Ed value of these crystals is not so high as compared to that of alloys. Herein, we investigated twinning deformation in a single crystal of 2-methyl-5-nitro-benzoic acid (C7H8NO4). Our results indicate exceptional mechanical twinning with an unprecedentedly large bending angle and large coercive stress, which requires drastic molecular rearrangement at the twinning interface to preserve the hydrogen bonding and avoid crystal cleavage. These factors contribute towards a large Ed, which is an important property for mechanical damping of vibrations.Recently, our research group has reported some stress-induced twinning deformation in organic single crystals,7H8NO4) (1) (2-Methyl-5-nitrobenzoic acid (CNO4) (1)  is a bei0α and (1̄1̄0)0α (mother domain: α0) crystal faces at 298 K. The crystal deformed in a diffusionless manner (0 and α1 (α1: twinned domain), which corresponds to the angle between (03̄1)0α and (031̄)1α, which agrees well with the value 64.28° obtained by measurement from the optical microscope s manner . The bencroscope . It is s0 and α1 crystal structures have an identical crystal structure with the same symmetry i.e. triclinic crystal system and P1̄ space group12 (Table S10α//(21̄1)1α. The α1 domain is related to α0 domain by a 180° rotation about the axis along [013]0α 2ClO4,14 (TMTSF)2PF6,14 3,5 difluorobenzoic acid,8 and 5-chloro-2-nitroaniline9 are 33.3%, 36%, 52.7% and 115.9%, respectively. Moreover, the strain value is higher than for certain twinned metals. For examples, the values for gold nanopillars15 and copper nanowires15 are 5% and 7.2%, respectively.The maximum strain of 1 is expected to be 218.81% from tanvia the crystal surface (110)0α , i.e. (13.40 × 10−15 m2) ) in the curve. This could be ascribed to the generation of the multiple domains. On holding the displacement of the jig, the α1 remained present and strain was recorded by removing the stress (iii and iv). The estimated dissipated strain energy was calculated as 1000 kJ m−3 . Based on the equation (Ed = σc tan θ), the Ed value of 1 is 65.24 times larger than the corresponding value for 3,5-difluorobenzoic acid i.e. 15.47 kJ m−3 ,8 and 4.67 times larger than in the case of 5-chloro-2-nitroaniline i e. 216 kJ m−3 .9 This large value of Ed is comparable to that of alloys and much higher than typical for organic solids. Since Ed is a derivative of the applied shear stress (σ) and deformation angle (θ) of the crystal, such dissipated energy density is expected to be increased by enlarging the bending angle of the crystal, which in this case is 64.28° α0 , which d0−15 m2) . One ends 64.28° , and/or Ed in organic single crystal, which typically bears smaller stress than those of metallic solids. Large bending angle in twinning ferroelastic deformation enables a wider crystal deformation range for the realization of large energy od dissipation. The relationship between effective bending angle and Ed can be clearly measured in single crystals without breakage by microscopic and macroscopic experiments. The maximum strain and the Ed value in 1 is, to the best of our knowledge, the largest reported molecular crystals, facilitated by large deformation but with a smaller shear force. Such controllability paves the way for new design principles to increase the value of Ed in organic single crystals. The combination of small σc and large Ed makes organic ferroelastic materials promising for applications in mechanical damping, with high susceptibility to absorb weak shocks effectively by their small volume.We confirmed a new strategy to increase There are no conflicts to declare.RA-008-C8RA02499E-s001RA-008-C8RA02499E-s002RA-008-C8RA02499E-s003


Citrus unshiu Marc. cv. Miyagawa-wase) is the most widely cultivated citrus variety in Korea. Although most varieties are imported from Japan, efforts have focused on developing new domestic varieties. We produced mutants by irradiating C. unshiu Marc. cv. Miyagawa-wase scions with gamma rays and grafting them onto C. unshiu Marc. cv. Miyagawa-wase branches. We compared the characteristics of these mutants with Miyagawa-wase fruit as a control. A mutant line named Jedae-unshiu with a unique fruit shape was selected for investigation in detail. The phenotype of Jedae-unshiu fruit demonstrated vertical troughs on the flavedo, smooth albedo without rough protruding fibers, and good adhesion between peel and flesh. In addition, Jedae-unshiu had thicker peels and higher fruit hardness than the control. Higher levels of hesperetin and narirutin, representative flavonoids, accumulated in the peel and flesh of Jedae-unshiu than those of the control. Cellular-level microscopic observations of the mature fruit peels demonstrated epidermal cell disruption in the control but not in Jedae-unshiu. Our results suggest that Jedae-unshiu has high possibility for development as a good storage variety containing large amounts of flavonoids, in addition to potential for ornamental value due to the unique shape of the fruit.Satsuma mandarin ( Citrus unshiu. Early-maturing satsuma mandarin (C. unshiu Marc. cv. Miyagawa-wase) is the most widely cultivated variety of citrus on Jeju Island. Miyagawa wase originated in Fukuoka Prefecture of Japan as a natural mutant of ordinary C. unshiu. This strain was later introduced into Korea by a French priest in 1911 and cultivated as a representative variety. C. unshiu Marc. var. Okitsu, the second-most cultivated citrus variety on Jeju Island, is derived from a branch mutation of C. unshiu Marc. cv. Miyagawa-wase. The cultivation area of C. unshiu Marc. cv. Miyagawa-wase accounts for more than 80% of the total citrus cultivation area on Jeju Island. Most produced citrus fruits are consumed as raw food, and non-commercial-sized fruits are processed into products, such as juice. It is necessary to develop a variety that can not only be consumed raw but also cultivated for ornamental purposes and processed with enhanced functionality :acetic acid [0.05%]) and B (acetonitrile [99.5%]:acetic acid [0.05%]) at a flow rate of 1 mL/min. The gradient profile was programmed at 30% B (0–28 min), 30–80% B (28–33 min), 80% B (33–38 min), 80–30% B (38–43 min), and 30–1% B (43–48 min). The column was equilibrated for 10 min with 30% B before the next injection. The sample injection volume was 10 µL, and flavonoids were detected at 280 nm. The results are expressed as the mean ± standard deviation of three replicates. All statistical analyses were carried out using IBM SPSS software . Significant differences among the samples were calculated using an analysis of variance followed by Duncan’s multiple range test at the 5% level  solution  for 24 h. The fixed tissue was dehydrated and made transparent using the following procedure: 50% ethanol for 5 h; solution I  for 5 h; solution II  for 37 h; solution III  for 5 h; solution IV  for 18 h; solution V  for 5 h; and solution VI  for 5 h. The dehydrated tissues were immediately placed into a carton and covered with paraffin, and the trimmed wax block was fixed on a microtome. The slice thickness was adjusted to 10 µm. The sections were stained as follows: xylene substitute for 60 min and 10 min; a mixture of 100% ethanol and xylene (1:1) for 10 min; 100%, 95%, 85%, and 75% ethanol for 10 min each; 1% safranin for 2 h; 75% ethanol for 5 min; 0.5% fast green for 30 s; 95% and 100% ethanol for 5 min each; a mixture of 100% ethanol and xylene (1:1) for 5 min; and xylene substitute for 10 min twice. The slices were examined and photographed using an optical microscope (Nikon microphoto type 114).The peels of young and mature


Piezoresponse force microscopy is used to study the velocity of the polarization domain wall in ultrathin ferroelectric barium titanate (BTO) films grown on strontium titanate (STO) substrates by molecular beam epitaxy. The electric field due to the cone of the atomic force microscope tip is demonstrated as the dominant electric field for domain expansion in thin films at lateral distances greater than about one tip diameter away from the tip. The velocity of the domain wall under the applied electric field by the tip in BTO for thin films (less than 40 nm) followed an expanding process given by Merz’s law. The material constants in a fit of the data to Merz’s law for very thin films are reported as about 4.2 KV/cm for the activation field, The online version contains supplementary material available at 10.1186/s11671-022-03688-2. There have been several recent studies of ferroelectric polarization domain formation under an applied electric field –15, manyv is the velocity of the lateral expansion of the domain wall; E, the applied electric field; U, the energy barrier between the initial and final polarization; k, the Boltzmann constant; T, the sample temperature; and The velocity of the lateral expansion of this domain wall is predicted by , 19, 21:t factor , 22, 23.While this behavior, expressed in Eqs. The experimental setup used to study domain formation and its lateral expansion, and an AFM tip are depicted in Fig. V is the applied voltage; a, the tip apex radius; r, the lateral distance from the tip–surface contact point; and d, the thickness of the film. In general, analytic approximations for the electric field in the film due to the AFM tip have been preferred over exact numerical simulations because they can be immediately used to verify Eqs. r, the lateral distance from the origin; V, the applied voltage; a , grown by a Riber 32 MBE, on strontium titanate doped with niobium  is found to be given as  was used to calculate the electric field in the BTO thin films between the conductive tip and substrate using the sphere–cone model for the tip. For this comparison, the same parameters for the AFM tip and for the material were used for both 1) the COMSOL simulation and (2) the corresponding analytical expressions for the electric field due to the cone Eq.  and Eq.  the COMSWe used PFM to quantitatively study the role of film thickness and applied voltage on the expansion of the polarization domain in ultrathin films. We (1) found the electric field due to the cone of an AFM tip is needed to explain the observed behavior of the lateral expansion of the polarization domain in thin films for radii larger than about one tip diameter away from the tip; (2) developed an analytic expression for the electric field due to the cone; (3) determined the dependence of domain expansion on applied voltage and on the thickness of the film; and (4) found that PFM data taken on BTO thin films agreed with Merz’s law with exponent Additional file 1. Supporting information for: The growth of polarization domains in ultrathin ferroelectric films seeded by the tip of an atomic force microscope.


Cajanus cajan (L.) Millsp.), a potential legume as an economical source of protein, is commonly cultivated in tropical and subtropical regions of the world. Therefore, pigeon pea may be potentially used as a substitute to improve the nutritional profile of foods. In the present study, the effect of substitution of whole wheat flour (WWF) with 20% and 40% pigeon pea flour (PPF) on the nutritional properties, color profiles, and starch and protein digestibility of chapati was investigated. The results showed that PPF had higher protein content but less carbohydrate than WWF. The protein content of chapati substituted with 20% and 40% PPF increased by 1.18 and 1.34 times, respectively, compared to WWF chapati, along with a marked decrease in carbohydrate content. Analyses further revealed an increase in the lightness and yellowness and a decrease in the redness of the chapati. Furthermore, glucose release from chapati with 20% and 40% PPF under simulated digestion was attenuated, corresponding to decreased hydrolysis and a predicted glycemic index. In the 40% PPF chapati, a significant reduction in slowly digestible starch (SDS) with increased resistant starch (RS) proportions was achieved without altering the effect on rapidly digestible starch (RDS). In addition, the level of amino-group residues was markedly elevated in 20% and 40% PPF substituted chapati compared to WWF chapati. These findings suggest that PPF can serve as a promising plant-based alternative ingredient to improve the nutrient value of chapati by reducing starch and increasing protein digestibility.Pigeon pea ( Plant-based alternatives have been increasingly recommended to minimize the consumption of animal-based foods . SeveralCajanus cajan (L.) Millsp. is a legume crop grown widely in Africa, Central America, and India [Pigeon pea, nd India . It has nd India . Variousnd India ,15. It ind India  and cracnd India . The comnd India . AlthougAspergillus niger was purchased from Roche Diagnostics . Glucose LiquiColor® was purchased from HUMAN GmbH .Pigeon pea was obtained from the local market, Tak, Thailand. Pepsin from porcine gastric mucosa powder, α-amylase Type VI-B from porcine pancreas, and pancreatin from porcine pancreas were obtained from Sigma-Aldrich Chemical . Amyloglucosidase from Pigeon pea was made into flour following a previous method, with slight modifications . BrieflyWhole wheat flour (WWF) was replaced by pigeon pea flour (PPF) at 20% and 40%. First, a dough was formed by mixing flour (100 g) with 60 mL of room temperature water using an electric mixer  for 5 min until a dough was formed . The finProtein, fat, total dietary fiber, moisture, and ash content in flour and chapati samples were determined by the Association of Official Analytical Chemists (AOAC) methods . Total cL* a* b* system. The total color difference (∆E) between WWF and PPF samples was calculated using the following formula [The color of chapati was measured using a colorimeter by the Hunter Lab Color Measuring System . The instrument was calibrated using the standard tiles. Portions of flour blends and chapati were randomly selected and homogenized using a grinder . Then, homogenous samples were placed in the sample holder, and the reflectivity was recorded in triplicate. The results were reported as an average and expressed according to the CIE  formula : ∆E = [. In brief, the working reagent (500 μL) was mixed with the sample digesta (5 μL) and incubated at room temperature for 10 min. The absorbance was measured at 500 nm using a microplate reader . Glucose (100 mg/dL) was used as a standard. The rate of starch digestibility was expressed as the glucose concentration at different time intervals . The glucose values (0–180 min) were plotted as a line graph, and areas under curves (AUCs) were calculated using the trapezoidal rule. The hydrolysis index (HI) was calculated according to the following equation:The glucose content in the digesta was measured using an enzymatic colorimetric GOPOD method (Glucose LiquiColorThe predicted glycemic indices (pGI) of the samples were estimated using the following equation ,25:pGI =Total starch was determined based on the method previously reported by Goni et al., with a slight modification . The sam0 denoted glucose released at time 0 min, G20 denoted glucose released at time 20 min, G120 denoted glucose released at time 120 min, and TS denoted total starch.The starch fraction was calculated according to the in vitro digestibility of the starch in the samples. The percentage of starch fraction was calculated based on the study of Englyst et al. ,28, wherDigesta were collected at the end of the gastric phase and in the intestinal phase at 0, 10, 20, 30, 40, 60, 90, 120, 150, and 180 min. The amino-group residue of samples was determined by the ninhydrin assay, with slight modification ,30. Brien = 3. The obtained values were statistically analyzed using one-way analysis of variance (ANOVA) using SPSS software . The significant difference was analyzed by Duncan’s multiple range tests at p < 0.05.The results were expressed as means and standard error of mean (S.E.M.), The proximate analysis of WWF and PPF is presented in a*) and an increase in yellowness (b*) were observed for PPF when compared to WWF. Furthermore, the lightness (L*) and yellowness of 40% PPF chapati were significantly higher than those of WWF chapati. The redness of the chapati was reduced only when increasing the substitution of PPF by 40%, suggesting that the color changes in chapati may be due to the effect of PPF substitution. An earlier investigation found a decreased lightness and an increased yellowness in chapati substituted with multigrain flours at 5–25% [E) between WWF and PPF was 1.30 ± 0.28. The total color difference from WWF chapati was 0.88 ± 0.44 and 1.92 ± 0.31 for chapati substituted with 20% and 40% PPF, respectively. As all ∆E values were in a range between 1 and 2, the results suggested that the color difference among samples could be detectable only by an experienced observer [The color attributes of flour in the chapati samples are presented in at 5–25% . As prevat 5–25% . Severalat 5–25% . Primariat 5–25% . Prior rat 5–25% . In the observer .In general, the pGI value of foods depends on the parameters of SDS and RS and the degree of gelatinization . TherefoThe amount of amino-group-containing compounds equivalent to lysine in the flour samples is shown in The food industry and current research exhibit an increasing trend in using plant proteins derived from grains, seeds, and legumes as substitutes for animal proteins . The plaThe development of chapati with whole wheat–pigeon pea flour blends could improve nutritional characteristics, while its color profile was relatively well maintained. Examining starch and protein digestibility together provides an overall understanding of how nutrient changes from pigeon pea flour substitution affect chapati digestion. A decrease in starch digestibility in chapati substituted pigeon pea flour was found, corresponding to an increment in resistant starch proportion. Digestible protein release from chapati was increased with the pigeon pea flour substitution, which could enhance protein absorption. These findings can be used as a starting point for further research on the usage of pigeon pea flour in wheat products. This calls for future investigation into the mechanistic formation of resistant starch, protein bioaccessibility, amino acid profile, and release during digestion to give a deeper insight. From the foregoing, pigeon pea can be considered as one of the plant-based protein alternatives that can be applied to foods to reduce starch digestibility and improve protein content.


Pumpkin seed flour is a promising raw material for use in the technology of various bakery products. It has a high biological value and valuable amino acid profile. During the technological process of making bread, there are conformational changes in the protein structure. The purpose of the study was to determine the effect of pumpkin seed flour on conformational changes in the structure of protein substances of dough and bread from wheat flour by near-infrared reflection spectroscopy. The protein profile changed to complete when replacing 10% or more of wheat flour because the score for all amino acids was higher than 100%. The utilitarian coefficient indicates the same balance of amino acids in proteins of all samples. As the percentage of substitution increases, the number of amino acids used for anabolic purposes decreases, and these are more fully utilized by the body. During recent years, the increasing interest of consumers in fitness regimes and a healthy body appearance contributed to a rise in the demand for healthy products. Furthermore, after the spread of COVID-19, consumers were forced to change their attitude toward food; by staying at home, a lot of people enhanced awareness about food waste and a healthy lifestyle, adopting a positive adaptive behavior related to healthy diets and food systems , modern Thus, among other recent trends, consumer attention to the nutraceutical aspect of food products is becoming mainstream, providing both challenges and opportunities to farmers and other actors involved in the agrifood supply chain: innovative “product-rich-in” and “free-from” became very profitable segments, offering, respectively, fortified/functional characteristics , and products with a low or no content of particular attributes such as fat, sugar, lactose, gluten, etc.Health problems can influence these food choices; however, an increasing number of people consume these products because of their linkage with a healthier lifestyle.Several studies stress the effect of cognitive factors, socio-economic status, food-related values, and traits that influence the interest of consumers in functional food ,4,5.In this context, the trend of using non-traditional raw materials to increase the nutritional value of products and provide them with healthy properties is rising in the bakery industry . DiffereIn addition to the nutritional components, in the case of pumpkin seeds, another aspect to consider is the reduction of agroindustry waste provided by reusing seeds flour in preparing bread, cake, and other foodstuffs. An important challenge of the food industry is using alternative plant parts, such as peels and seeds, with a significant nutritional value in the formulation of food products, enriching their functional value and reducing costs in waste treatment . Indeed,The addition of pumpkin seed flour in the amount of 5–15% to whole wheat flour in the technology of bread increased crude protein, ash, and crude fat content, but the addition of higher levels of pumpkin seed flour might cause rancidity .The size of the particles of pumpkin seed flour is larger than wheat wholemeal flour, and the water absorption capacity exceeds such an indicator for wheat flour by 1.5 times . The gasThe increased amount of added partially defatted pumpkin seed flour 5–15%) decreased the water absorption capacity of the dough, and the dough stability increased from 9.43 to 10.23 min [% decreasA key role in forming the structure of the dough belongs to the gliadin and glutenin fractions of protein, which, in the hydrated state, form gluten, a continuous viscoelastic network. In the technological process of making bread, there are molecular conformations of gluten proteins altering the viscoelastic properties of dough systems. They can be evaluated by numerous techniques, e.g., infrared spectroscopy .Glutenins are multi-chained proteins, polymerized by disulfide bonds and not cross-linked. They provide gluten with elastic properties. Gliadin proteins contain intra-molecular disulfide bonds . The breThe amino acid profile and protein digestibility in vitro provide the quality of proteins. The in vitro protein digestibility of pumpkin seed kernels is 90%, and that of defatted pumpkin seed flour is 77.91% .In dough, there are microstructural and molecular changes in gluten proteins. The amorphous matrix of gluten at the microstructural level experienced a different degree of depolymerization into laminar and fibrillar structures. The development of these elements was the result of conformational changes in the secondary structure of gluten proteins . BecauseThe aim of the study was to determine the effect of pumpkin seed flour on conformational changes in the structure of protein substances of dough and bread from wheat flour.Flour plays a decisive role in forming the properties of dough for bakery products, especially for its protein content and composition . The totIndeed, through the analysis of the fractional composition of proteins, higher albumin, glutenin, prolamin, and insoluble protein content in wheat flour was observed when compared with the pumpkin seed flour . A higheThe highest water-soluble and salt-soluble protein fractions found in the pumpkin seed flour can affect chemical processes in the dough , and its structural and mechanical characteristics .The high biological value of pumpkin seed flour is due to a higher presence of EAA than that found in the wheat flour .The analysis of EAA showed a significantly higher EAA concentration in pumpkin seed flour when compared with the wheat flour. The predominant essential amino acid of pumpkin seed flour was leucine, whereas tryptophan was the lowest. This composition was confirmed by other authors , even thThe functional and technological properties of flours determine their behavior during processing, and characterize the ability to bind and retain moisture . Indeed,Both moisture binding capacity and moisture retaining capacity were significantly higher in pumpkin seed flour than in wheat flour due to the higher content of fiber generally present in pumpkin seed . This asFlour is a material which consists of a set of particles. Their size exceeds the wavelength of light. Therefore, there is a propagation of optical radiation in all directions , which forms a white reflection. To identify and analyze the components of such materials, it is advisable to use the reflection spectrum in the near infrared area . The refAfter kneading the dough, the spectra of the obtained samples with different concentrations of pumpkin seed flour to replace part of the wheat flour differed slightly, even though all the extremes were identical . HoweverThe total time of fermentation and keeping of the dough is 3.5 h. The conformational changes in the structural substances of the dough were monitored after the completion of the fermentation . After kComparing the control dough sample analyzed immediately after kneading  and afteDuring the process of baking, under the influence of high temperatures (220 °C), changes in protein and other substances of the product occurred, uniforming the reflection intensity of the final products . Indeed,A variety of functional groups  can be quickly and reliably identified using infrared spectroscopy. In particular, most food products contain OH, CH, and NH functional groups, which are determined by the formed spectra . Spectra arising from the oscillations of various groups in the near-infrared region have characteristic bands and characteristic frequencies . In the Analyzing the reflection spectra of the dough after kneading, after 3.5 h of fermentation, and of bread (both control sample and sample with 20% pumpkin seed flour), the highest reflection was noted at wavelengths of 1460 nm, 1760 nm, 1930 nm, 2100 nm, 2270 nm, and 2350 nm , with thThe evaluation of the protein content in food products is usually carried out at 1510 nm , 2060 nm , and 2180 nm ; however, these spectra did not show any extreme reflection.The spectrum of the control sample of bread was the highest among all samples, except at 1930 nm, where the control sample of dough after 3.5 h of fermentation reported a reflection intensity of 0.5 lg 1/R. Bread with 20% pumpkin flour had a slightly lower intensity than the control bread, but, in some areas, both the spectra were overlapping, in particular at 1730, 2050, and 2250 nm. The other three spectra  were located in a separate group with lower reflection intensity when compared with bread samples and control dough after fermentation. The spectra of the dough with 20% pumpkin seed flour had the lowest reflection intensity among all samples, but the sample after kneading was located above the fermented sample. Regardless of the type of used flour, the samples of bread had a higher intensity of reflection than dough. This can be explained by the effect of high temperature in the baking process, which causes significant changes in protein and starch, fat, and other substances . Indeed,The EAA profile of bread with different percentages of pumpkin seed flour is reported in Finally, the utilitarian coefficient of the EAA composition, characterizing the balance of EAA in relation to the physiologically necessary norm, was calculated . To charAnalysis of the utilitarian coefficient, redundancy coefficient, and biological value of bread enriched with different percentages of pumpkin seed flour indicates a similar balance of EAA in proteic structures in all samples. However, as the percentage of pumpkin seed flour enrichment increased, the DCAS decreased, subsequently increasing the use of EAA by the human body, which was dependent on the pumpkin seed flour percentage.Protein composition and functional and technological properties  of premium wheat flour and pumpkin seed flour were analyzed following the methods described in paragraphs 2.2–2.5 and compared. During the flour preparation, 500 g seeds of wheat and of pumpkin bought in a local market were milled in a heavy grinder, sifted through a mesh sieve (200 μm aperture size) to obtain a fine powder, and kept at 4 °C until further use. Then, dough samples were prepared, each with 200 g premium wheat flour, 5 g salt, 7 g pressed baker’s yeast, 300 g water, and pumpkin seed flour in the amount of 5%, 10%, 15%, and 20% to replace wheat flour, and the prepared dough samples were fermented for 3.5 h at room temperature (25 °C). A sample without pumpkin seed flour was used as a control sample. After the investigation of semi-finished products, bread from doughs prepared with different concentrations of pumpkin seed flour and wheat flour for the control sample was prepared, cooking at 220 °C for 25 min in a ventilated oven . Three breads for each treatment (each from 500 g of dough) were obtained and stored at room temperature (25 °C) for one week, until the analysis. Flours were analyzed for total protein content, mass functional composition of protein, essential amino acid profile, and their biological and technological value. The reflection spectroscopic profile of doughs was analyzed immediately after kneading and after 3.5 h of fermentation. Finally, the bread was analyzed spectroscopically for its EAA composition and biological value.2O was added to the hydrolysates before neutralization and titration [Flour mineralization with a strong acid releasing nitrogen can determine N content by the titration technique, following the Kjeldahl method. An amount of 1 g of raw material was hydrolyzed with 15 mL concentrated sulfuric acid containing two copper catalyst tablets for 2 h in a heat block at 420 °C. After cooling, distilled Hw/v ratio for 24 h, and the solvent was then removed by centrifugation. The mixture was dried at room temperature and stored in airtight sample bottles at 4 °C until subsequent use. Then, pumpkin proteins were fractionated from pentane defatted meal according to the Osborne differential extraction procedure described by Horax et al. [g to separate the supernatant from the pellet, thus obtaining the deionized water extract (DWE). The same extraction/separation conditions were kept for the next successive protein extraction steps. The water extract pellet was resuspended into 100 mL of 1 M NaCl solution and stirred as mentioned above. The supernatant obtained after centrifugation, designated as salt extract (SE), was then extracted in 100 mL of deionized water adjusted at pH 11 with 0.5 M NaOH solution, leading to the alkaline extract (AE). Each extraction was repeated twice. After each extraction, the pellets were washed twice using 20 mL of solvent to collect the residual protein entrapped in the insoluble residues. The DWE, SE, and AE were precipitated for isolation by adjusting the pH of the obtained supernatant to the pH corresponding to the minimum of solubility (pHms) determined from the turbidity experiment [g for 15 min, the isolated protein precipitates were washed twice using deionized water at their respective pHms and recentrifuged. Finally, the resulting protein fractions were resolubilized by adjusting the pH to 7.0, freeze-dried, and stored at 4 °C until further analysis [The seed flour was defatted with pentane. The flour/solvent slurry was stirred at a 1:10 x et al. . The meaperiment . The pH analysis .iM is molecular mass of each amino acid; iS is an area of peak of each amino acid on an aminogram from the investigated solution; eS is an area of peak of each amino acid on an aminogram from the solution of the standard mixture of amino acids which accords to one micromole.The determination of amino acid composition was conducted in accordance with the method of ion exchange chromatography . The quaAmino acid SCORE is expected according to the certificate scale of THEO/WHO .Moisture binding capacity was determined by the centrifugation method , and wasThe determination of the moisture retaining capacity was carried out by mixing the sample with water and determining the amount of separated liquid after a centrifugation , and wasThe reflection spectra from a smooth surface and shredded samples were researched by using an Infrapid spectrometer  in the near-infrared range from 1330 to 2370 nm. The spectrometer initially recorded the reflectance spectrum from reference I0 (component part of the instrument); then, a reflection spectrum from the researched sample I was obtained. The spectra are represented as the reflectivity of R in relative units (the ratio of the intensities I/I0 = R), depending on the wavelength in nm ,50,51. TminC is minC—score of the first limited acid EAA units; jA is the mass fraction of the j-th of EAA in the product, mg/g protein; ejA is the mass fraction of the j-th of EAA in the protein model, mg/g protein, according to FAO/WHO scale.The utilitarian coefficient (U) characterizes the balance of all EAA proteins with respect to the physiological norm. It is used to compare the protein composition of various food products based on their amino acid composition and the inadequacy of their use in the body. It was calculated by the formula:red) shows the mass fraction of EAA in 100 g of the product, which is not fully used by the body. It was calculated by the formula:The redundancy coefficient , expressed as % , was calt-test using a significance level of 0.05. The results of the analyses of different breads were subjected to a one-way analysis of variance (ANOVA), using the different concentrations of pumpkin seed flour  as the factor of variation. All the means were separated by an LSD (Least Significant Difference) post-hoc test (p < 0.05). The normality of data was tested using the Shapiro–Wilk test, whereas the homoscedasticity was tested using Bartlett’s test. Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad .The results of the analyses of different flours (from wheat and from pumpkin seed) were compared with a two-tailed Student’s Conformational changes in the structure of protein substances of dough and bread from wheat flour occurred when adding pumpkin seed flour, due to their different protein compositions. The total content of proteins found in pumpkin seed flour was 40% lower than that of wheat flour, with a higher presence of EAA.Near-infrared reflection spectroscopy confirmed the difference in the fractional composition of proteins of the studied flours, and the different content between soluble and insoluble proteins, as well as the presence of OH, CH, SH, and NH groups.The protein profile was complete after the replacement of 10% or more of wheat flour with pumpkin seed flour; this could be because the score for all amino acids was higher than 100%. As the percentage of the substitution of wheat flour with pumpkin seed flour increases, the number of amino acids used for anabolic purposes decreases, and they are fully utilized by the human body. Further research must be conducted in order to determine the best content of pumpkin seed flour to introduce in wheat bread. Concerning further research to be carried out in light of the results of this research, taking into consideration the variations that different cultivars and phenotypes of pumpkin may have in terms of production and nutritional characteristics , from a Furthermore, sensory acceptance analysis might explore the level of preference and the purchase intention of consumers in order to position pumpkin seed flour products in the right target market.


We observed that the G228W mutation in the SAND domain negatively influences the AIRE-SIRT1 interaction. The disturbed interaction might cause a disruption in the binding of the AIRE SAND domain with the SIRT1 catalytic site, impairing the AIRE complex to proceed downstream with RNA Pol II.The autoimmune regulator (AIRE) protein functions as a tetramer, interacting with partner proteins to form the “AIRE complex,” which relieves RNA Pol II stalling in the chromatin of medullary thymic epithelial cells (mTECs). AIRE is the primary mTEC transcriptional controller, promoting the expression of a large set of peripheral tissue antigen genes implicated in the negative selection of self-reactive thymocytes. Under normal conditions, the SIRT1 protein temporarily interacts with AIRE and deacetylates K residues of the AIRE SAND domain. Once the AIRE SAND domain is deacetylated, the binding with SIRT1 is undone, allowing the AIRE complex to proceed downstream with the RNA Pol II to the elongation phase of transcription. Considering that the  AIRE) gene spans 14 exons at chromosome 21q22.3, encoding a 1635-nucleotide mRNA and a 545-amino acid protein (AIRE gene (https://www.hgmd.cf.ac.uk/ac/). Some mutations in the AIRE gene are related to autoimmune polyendocrinopathy-candidiasis ectodermal dystrophy (APECED) (The human autoimmune regulator ((APECED)  and a G2(APECED) –10.The 56-kDa AIRE protein is a transcriptional regulator in medullary thymic epithelial cells –14, exhiIn addition to chromatin binding, the SAND domain may also have a regulatory role in the homomultimerization of AIRE , 24. TheConsidering that i) the deacetylation of SAND domain K residues mediated by the partner SIRT1 molecule is essential for AIRE function in releasing RNA Pol II and allowing it to proceed with the elongation phase of transcription , 25–28, in silico analyses showed differences in the physical interaction between the AIRE SAND domain and the SIRT1 catalytic site for both wild-type and mutant domains. On the other hand, the surface plasmon resonance evaluation showed a stronger affinity (as seen through binding and dissociation) of the wild-type SAND domain peptide with the SIRT1 protein compared to a weaker association observed for the mutant peptide.We integrated computational molecular modeling, docking, and dynamics between the whole AIRE SAND domain with the SIRT1 protein to test this hypothesis. Additionally, we evaluated the binding of a peptide harboring the 211 to 230 residues of the SAND domain with the SIRT1 protein to compare the binding/release between AIRE G228 (wild-type peptide) and W228 (mutant peptide) using surface plasmon resonance. The http://swissmodel.expasy.org/workspace). The amino acid sequence in the FASTA format of the SIRT1 target protein was used as input. The SWISS-MODEL algorithms performed BLASTp and provided the best homology models based on global model quality estimation (GMQE) and qualitative model energy analysis (QMEAN) statistical parameters. The GMQE provided the quality by combining the target-template alignment properties, estimating a range between 0 and 1. The scoring function of QMEAN consisted of a linear combination of structural descriptors, which is related to the high-resolution X-ray crystal structure, estimating a Z-score scheme.We used the SWISS-MODEL workspace for protein modeling (https://cluspro.org/) was used. In the ClusPro server, the rigid body docking phase uses the PIPER docking program (https://www.schrodinger.com/products/piper), which relies on the fast Fourier transform (FFT) correlation approach. PIPER represents the interaction energy between two proteins using the formula E = w1Erep+w2Eattr+w3Eelec+w4EDARS, where E rep and E attr denote the attractive and repulsive contributions to the van der Waals interaction energy, and E elec represents the electrostatic energy. EDARS is a pairwise structure-based potential; it primarily means desolvation contributions, i.e., free energy change by removing the water molecules from the interface. The coefficients w 1, w 2, w 3, and w 4 define the molecular weights of the corresponding amino acid residues.Molecular docking analysis of the modeled AIRE SAND domain, wild-type or G228W mutant AIRE SAND domain, and the SIRT1 protein (PDB code 4I5I) was performed to study the stability of the protein-protein interaction. The ClusPro 2.0 protein-protein docking server   structures of the complexes formed with AIRE wild-type or with AIRE mutant peptide and SIRT1 generated in the docking section were used as initial configurations for the molecular dynamics simulations. In this work, calculations were performed to verify the stability and affinity of the complexes.https://ambermd.org/) for this residue. ACK and the connectivity library file for K222 residue were generated by tleap.To mimic the interaction between the two peptides and SIRT1 protein, the K222 residue was acetylated using the parameters given in the AMBER Molecular Dynamics Package (https://ambermd.org/AmberTools.php). The whole system was truncated in a box with 15 Å between the edges of the periodic box and the molecules analyzed. An 8 Å cutoff particle mesh Ewald (PME) method was used to treat the long-range electrostatic interactions. AMBER molecular dynamics software and the ff19SB force field were used to prepare the systems and run the simulations.The two systems were solvated using the TIP3P water model and neutralized with four Na+ ions in the tleap tool implemented in AMBERTOOLS21  (https:/https://ambermd.org/) to constrain hydrogen bonds.Before running, the complexes were subjected to minimization in two stages using the steepest descent method followed by a conjugate gradient. In the first stage, the elements, peptides, and SIRT1 were fixed, and the position of the water molecules was minimized. After that, the whole system was minimized. A restrained thermalization phase with the microcanonical ensemble (NVE) was performed, increasing the temperature by 0.1 K until it reached 300 K using the Langevin thermostat and SHAKE algorithm (https://pymol.org/2/).The production dynamics were carried out at constant pressure (NPT ensemble) with a time step of 2 fs. The total simulation time for each complex was 2700 ns, and frames were saved every 20 ps. The analyses were performed in the last 2650 ns after the systems were equilibrated. All calculations shown in the current work were performed using AMBERTOOLS21 scripts. The visualization programs used to create the figures of the molecules were VMD for LINUXAMD64, version 1.9.3,  and PYMOThe surface plasmon resonance (SPR) experiments were performed on Biacore Model T200 equipment . For the immobilization of human SIRT1 , a CM5 sensor chip S series  has carboxylic groups available for covalent coupling was used. All solutions used were purchased from the equipment supplier . The procedures described here were performed automatically with the Biacore instrument using pre-established programs for binding amines in peptides or proteins.Briefly, the carboxylic groups on the CM5 sensor chip surface were activated with EDC + NHS, forming the NHS ester reactive intermediate, which, after washing with HBS-N, readily reacted with amines dissolved in the immobilization solution. The human SIRT1 ligand was dissolved in 10 mM sodium acetate pH 4.0 plus 20 µM NAD+ , which was immobilized onto the Biacore sensor chip according to the manufacturer´s instructions.To detect interactions between SIRT1 and AIRE WT or AIRE G228W mutant SAND domain peptides, different concentrations of peptides were injected onto the CM5 chip with immobilized SIRT1 protein. Peptide samples were injected sequentially from lowest to highest concentrations  with a contact time of 300 s, a 20 μl/min flow, and a dissociation time of 1200 s. The surface was regenerated between samples by injecting 2.0 M glycine pH 2.0  followed by injection of 2.0 M NaCl . The stabilization period between injections after regeneration was 200 s.KCIQVGGEF) and AIRE G228W mutant (IQQVFESGGSKKCIQVGWEF). The underlined K residue represents the position where the acetyl radical was added. The experiment determined whether the K residue underlined was acetylated or not. The AIRE wild-type or AIRE G228W mutation data are available through GenBank NCBI (https://www.ncbi.nlm.nih.gov/) accession numbers (NM_000383.4): c.682G>T G [GGG] > W [TGG], coding sequence variant autoimmune regulator (NP_000374.1): p. Gly228TrpG (Gly) > W missense variant, DbSNP (https://www.ncbi.nlm.nih.gov/snp/rs121434257) and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/variation/3313/).The respective sequences of the twenty-amino acid peptide residues  comprising positions 211 to 230 of the SAND domain of the human AIRE protein are as follows: from the C to N-terminus, AIRE wild-type (IQQVFESGGSKwww.skscience.org/anabel). Data obtained with the different concentrations of AIRE WT or mutant peptides were combined in the same graph.Kinetic data were analyzed with Anabel Analysis of Binding Events software , availabTo obtain a separate 3D human AIRE SAND domain structure, we used the SwissModel tool to model proteins through homology. We used the SAND domain sequence of the human SP100 protein (PDB code 1h5p) for the modeling, which features 39.62% sequence identity to the human AIRE SAND domain. The SP100 protein SAND domain conserves the SKKCIQ amino acid sequence in the AIRE SAND domain, corresponding to the accurate interaction site between SAND and the SIRT1 protein. Thus, obtaining a 3D structure of the wild-type AIRE SAND domain and the AIRE SAND domain carrying the G228W mutation was possible. Considering that the G228W mutation is located in a loop region of the AIRE SAND domain Figure 1To obtain an interaction model between the AIRE SAND domain (wild-type or G228W mutant) and SIRT1 and assess the affinity of the mutant SAND domain, we used the ClusPro 2.0 blind molecular docking tool. The docking was performed with nonacetylated K222 to understand whether the modeled AIRE SAND domain would have an affinity for the catalytic region of the SIRT1 protein.Through blind molecular docking, it was possible to observe an affinity between the AIRE SAND domain WT G228 or W228 mutant domain and the catalytic region/site (H363 residue) of the SIRT1 protein, even without any previous targeting or chemical modification of the K222 residue, which is a target of the SIRT1 protein Figure 2Comparing the structural conformations, we observed that the SAND domain mobilizes the W228 mutant residue that interacts with the SIRT1 H363 catalytic residue in the docking analyses Figure 3The molecular dynamics results with the AIRE SAND domain (wild-type or G228W mutant) showed that even the acetylated K222 residue at 1 µs of simulation did not allow the wild-type domain to form a stable structure, in which the K222 residue would interact with the H363 residue from the catalytic site of SIRT1. On the other hand, the G228W mutant domain exhibited a stable interaction with the SIRT1 catalytic site at the H363 residue. The analysis of the dynamics was performed to understand if K222 acetylation would be able to direct it to the catalytic residue of SIRT1.The RMSD results showed that the WT SAND domain and SIRT1 complex is slightly less stable along the simulation time (1200 ns) than the complex formed between the SAND G228W mutant. Nevertheless, the G228W mutant domain complex remained stable in the RMSD [in silico analyses, show that the G228W mutation changes the SAND-SIRT1 interaction, which is necessary to activate the AIRE protein. Of note, SPR results were in contrast with the blind docking and molecular dynamics, i.e., instead, the WT complex was more stable and mutant less stable. The SPR results were reproduced with six different concentrations of each of the acetylated peptides, WT and mutant (range 10 to 200 µM), in addition to the control (0 µM)  to study the behavior of the two types of peptides when they bind to SIRT1. The SPR results indicated a differential association and dissociation dynamics between the wild-type SAND G228 or mutant SAND W228 peptides and the SIRT1 protein. The interaction kinetics showed that the wild-type SAND G228 peptide has a more significant association capacity as observed at a concentration range of 20 to 200 µM. The interaction between the wild-type G228 SAND peptide and SIRT1 was almost completely dissociated after 1200 seconds of measurement. Conversely, the mutant W228 SAND peptide was less able to associate with SIRT1 Figure 6We reported that the dominant G228W mutation observed at the SAND domain of the human AIRE protein found in APS-1 patients . The binThe study of the molecular mechanisms of primary immunodeficiencies associated with the development of autoimmune disorders has contributed to understanding the mechanisms of tolerance loss to self-antigens. Although recessive AIRE mutations at the PHD1 domain may occur at a ratio of 1:1000 in the general population , AIRE SAThis study evaluated whether the G228W mutation changes the interaction with SIRT1, an essential partner protein of the AIRE complex. We accessed the 3D structure for the AIRE SAND domain by homology modeling to the human SP100 protein SAND domain, which is available in a public database. We observed that the G228W replacement, which occurs in a loop region, might not result in misfolding of the AIRE SAND 3D structure since loop regions do not present secondary structures , 34. TheFor this purpose, the structures of the wild-type SAND domain, the G228W mutant output from homology modeling, and the structure of the SIRT1 catalytic domain (PDB code 4I5I) were used. With the domains modeled, we used blind docking to understand how the complex between the SAND domains of AIRE WT and mutant would be formed with the SIRT1 protein.We observed that the SAND domain mobilizes the W228 mutant residue that interacts with the SIRT1 H363 catalytic residue, changing with the interaction kinetics between these proteins, followed by SPR measurements. As observed in this study, due to the SAND G228W mutation, the AIRE protein might bind weakly to SIRT1. The AIRE complex, including RNA Pol II, formed with an AIRE mutant protein, might be dysfunctional and keep stalled to chromatin. The dysfunctional AIRE complex might occur due to the non-deacetylation of K222 residue, which is essential for the proper function of the AIRE protein. In addition, to signify a non-physiological situation, this finding might explain the accumulation of the AIRE complex in the nucleus of mutant cells, as previously reported , 24.The AIRE protein plays a role in the cell nucleus as a tetramer , 19, in Although there is a prediction that the K222 residue is only moderately acetylated , previouin vitro tool used for determining binding kinetics between biological molecules, including antigen-antibody or protein-protein interactions, among other molecular interactions  (The complementary approaches used in this study (and SPR) , 40 alloThe original contributions presented in the study are included in the article/JS: raised hypothesis, discussed the analysis tools, performed molecular docking, analyzed data, interpreted the results, and wrote the manuscript. MD: discussed the analysis tools, performed molecular dynamics, analyzed data and interpreted the results. AM: performed protein binding kinetics (SPR), and interpreted the results. VF: performed protein binding kinetics (SPR), and interpreted the results. RB: performed molecular dynamics, analyzed data. ED: raised hypothesis, interpreted the results and wrote the manuscript. GP: conceived the study, raised hypothesis, organized the study pipeline, interpreted the results and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was funded by the following Brazilian research support agencies; Fundação de Amparo à Pesquisa do Estado de São Paulo , Conselho Nacional de Desenvolvimento Científico e Tecnológico , and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.