The synthesis, acid-base equilibrium, aggregation properties, and antibacterial activity were investigated. Conductivity and fluorescence were used to establish critical micelle concentrations. Micellization of CnPC3NH3Cl and CnTC3NH3Cl occurred in the ranges of 0.42–16.2 mM and 0.29–4.6 mM, respectively. Since those surfactants have some acidic character, the apparent pKa was determined through titrations, observing increasing acidity with increasing chain length and being slightly more acidic with the phenylalanine than the tryptophan derivatives. Both families showed promising antibacterial efficacy against eight different bacterial strains. Molecular docking studies against the enzyme peptidoglycan glycosyltransferase (PDB ID:2OQO) were used to investigate the potential binding mechanism of target surfactant molecules. According to small angle X-ray scattering (SAXS) results, the surfactants incorporate into DPPC  bilayers without strong perturbation up to high surfactant concentration. Some of the C12TC3NH3Cl/DPPC formulations (40%/60% and 20%/80% molar ratios) exhibited good antibacterial activity, while the others were not effective against the tested bacteria. The strong affinity between DPPC and surfactant molecules, as determined by the DFT  method, could be one of the reasons for the loss of antibacterial activity of these cationic surfactants when they are incorporated in vesicles. Cationic surfactants based on phenylalanine (C Infectious illnesses are a major problem for both human and animal health, along with the macroeconomy implications . The fasIt has been shown that traditional antimicrobials rapidly lose efficiency owing to microbial resistance, and biocidal diffusion can cause environmental pollution and human toxicity . VancomySurfactants and detergents, which remove debris from surfaces, are advised as a component of medical cleaning and sterilization procedures to perform in tandem with some other antibacterial compounds . In addiIn this context, amino acid-based surfactants represent a promising starting point in the development of new green and sustainable antimicrobial materials. These are structurally heterogeneous amphiphilic compounds with an amino acid hydrophilic group and an aliphatic chain as the lipophilic moiety. Due to their amphiphilic nature, positive charge, and high surface activity, these cationic amphiphiles, like QACs (Quaternary Ammonium Compounds), are expected to act via perturbation of the bacterial membrane ,14,15. TGreen amino acid surfactants are promising candidates for preparing cationic vesicles. These vesicles are interesting aggregates that can be used in numerous biomedical applications . They caIn this work, we have designed and synthesized two new families of cationic phenylalanine and tryptophan-based surfactants that can be prepared using a synthetic procedure that agree with the green chemistry principles. The chemical structure of these new surfactants was planned considering the structural requirements for readily biodegradable surfactants . The mic® Medium 712: PYG w/Additives was from Manassas. All solvents were reagent grade and used without further purification. Octanoyl chloride, Caproyl chloride, Lauryl chloride, and Myristoyl chloride were obtained from Fluka, and amino acids from TCI Europe. Trifluoroacetic acid (TFA) and dimethylformamide (DMF) were supplied by Merck. The culture media Sabouraud dextrose agar and CHROMagar were acquired from Himedia . The ATCCL-phenylalanine methyl ester or L-tryptophan methyl ester  were dissolved in 250 mL of acetone/water (34/60). The pH of the mixture was adjusted to 10 using an aqueous NaOH solution (1 M). After, 6.12 g (1.3 × 0.02 mol) of alkanoyl chloride  was added dropwise, and the mixture was stirred at room temperature for 4 h. The solid product obtained was filtered, washed with a solution of acetone/water at pH = 10, and dried using freeze-drying. Pure compounds were obtained by several crystallizations from hot acetonitrile. These intermediates were identified using HPLC, MS spectroscopy, and NMR.nPOM or CnTOM  was weighed in a round flask and 2.06 g (20 mmol) of 1,3-diaminopropane was added. The reaction mixture was maintained at 60 °C for 2 h. CnPNH2 and CnTNH2 were obtained by removing diamine under reduced pressure. Then, CnPNH2 or CnTNH2 were reacted with HCl in a methanol medium to obtain the target CnTC3NH3Cl and CnPC3NH3Cl. The reaction mixture was maintained at room temperature for 30 min, the excess HCl was removed with a rotary evaporator, and the sample was dried overnight under freeze-drying. The target surfactants were identified using HPLC, MS, and NMR  of TFA in H2O/CH3CN (1:4).The reaction progress, as well as the purity of all prepared molecules, were monitored with an HPLC (Merck-Hitachi D-2500) using a UV–vis detector (L- 4250 at 215 nm) and a Lichrospher 100 CN. The gradient elution profile used went from 25 %B to 95 %B in 24 min. Aqueous phase A was 0.1% (vol/vol) trifluoro acetic acid (TFA) in H1H NMR and 13C NMR analyses were recorded on a Varian 400 MHz spectrometer. All experiments were performed in an indirect broadband probe. Data were processed with the MestReNova (Mestrelab Research 14.1) software.Sample solutions were prepared in deuterated methanol (20 mg/0.8 mL) using 5 mm NMR tubes. −4 to 1 × 10−6 M range) were prepared in methanol. High-resolution mass spectra (HRMS) were performed on Acquity UPLC System and an LCT PremierTM XE Benchtop orthogonal acceleration time-of-flight  equipped with an electrospray ionization source. All data were processed and displayed using MestReNova (Mestrelab Research 14.1) software.Surfactant solutions  in the emission spectra of pyrene depends on the environment polarity and can be used to calculate the CMC [Steady-state fluorescence measurements of pyrene in surfactant solutions were performed at 25.0 ± 0.1 °C  using a 1 cm path-length quartz cuvette. From a stock solution of pure surfactants prepared in a pyrene-deionized aqueous solution of 10 the CMC .−1. Samples for conductivity measurements were prepared in Millipore ultra-pure water . Measurements were made at increasing concentrations to reduce errors from possible electrode contamination. The conductivity of water was subtracted from the measured conductivity of each sample. All measurements were performed in triplicate.Conductivity experiments were performed at 25.0 ± 0.1 °C using a Thermo Orion 5 Star multiparameter instrument  equipped with an Orion conductivity cell 913005MD with an epoxy/graphite electrode and a cell constant of 0.475 cma values, back-titration was also performed. In the cases where both procedures show an equivalence point, the obtained values for pKa were coincident within 0.3 units in the worst case. pH was measured using a pH electrode model 8103BN ROSS semi-micro ThermoOrion  connected to a DualStar ThermoOrion pH-meter. Titrations were performed under a nitrogen gas atmosphere with constant magnetic stirring. The apparent pKa was determined based on the pH corresponding to the semi-equivalence point of the neutralization curve.The apparent pKa values of the tryptophan and phenylalanine-based surfactants were determined by titration of about 30 µmol of aqueous surfactant solution at around 2 mM concentration with 20 mM aqueous sodium hydroxide at 25 °C. Because of high pKα line with more than 97% purity and less than 0.3% Kβ. A position X-ray detector was used in a transmission configuration . The temperature was controlled by means of a TCCS-3 Peltier , and the scattering patterns were recorded at 25 °C. The dispersions were loaded in a flow-through glass capillary of 1 mm diameter and 10 μm wall thickness . The SAXS scattering curves are shown as a function of the scattering vector modulus, according to the following formula equation (Equation (1)):θ is the scattering angle and λ the wavelength. The q values with this setup ranged from 0.2 to 6 nm−1. The scattering vector was calibrated by measuring a standard silver behenate sample. Scattering was accumulated in 20 min files for a total of 2 h. Once verified that there is no drift in the measurements, those spectra are summed and the resultant is background subtracted (water in this case) and scaled to absolute intensity by comparison with the water intensity. SAXS patterns were analyzed using a home-made fitting procedure based on a Gaussian description of the bilayers and using a Levenberg-Marquardt minimization scheme [Small-Angle X-ray Scattering (SAXS) measurements were used to gain structural information on the aggregation of the surfactants and their mixtures with phospholipids. Measurements were carried out using an S3-MICRO  coupled to a GENIX-Fox 3D X-ray source  working at 50 kV and 1 mA. The detector-focused X-ray beam is produced by a cooper anode with λ = 0.1542 nm Cu Kn scheme ,24,25,26Bacillus subtillis (ATCC 6633), Staphylococcus epidermidis (ATTC 12228), Staphylococcus aureus (ATCC 6538), Listeria manocytogenes (ATCC 153131), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 8739), Acinetobacter baumannii (ATCC 19606), and Klebsiella aerogenes (ATCC 13048). These bacteria were kept and grown following the guidelines set out by the National Council for Clinical Laboratory Standards [−5 colony-forming units (CFU) per mL. Nutrient broth medium without the compound served as growth control. The development of turbidity in an inoculated medium is a function of growth and reflects an increase in both mass and cell number. The MIC was defined as the lowest concentration of antibacterial agent that inhibited the development of visible growth after 24 h of incubation at 37 °C. To confirm this observation, 20 μL of resazurin at 0.015% w/v was added to each well and left to react for approximately 1 h at 35 °C. After the incubation period, the indication of bacterial growth, i.e., changing from blue to pink, confirmed the MIC value. To obtain the MBC, the antimicrobial concentration corresponding to at least 3 log reductions of viable cells, an aliquot of 10 μL of the MIC well, and the 2 concentrations immediately above were seeded over agar MH and incubated for 24 h at 35 °C. The MBC was determined as the lowest concentration in which no colonies were observed on the agar plates.Serial dilutions of the surfactant solutions and their vesicles were evaluated for antibacterial activity. The bacterial strains utilized were: tandards . The min ATTC 122, Staphyl9 cells/mL. For the preparation of erythrocyte suspensions, fresh blood was taken from a rabbit using a procedure approved by the institutional ethics committee on animal experimentation. The erythrocytes were washed three times in PBS (pH 7.4). Finally, erythrocytes were suspended in PBS at a cell density of 8 × 1050).To determine the hemolytic activity of the catanionic mixtures prepared in this work, we adapted the procedure described by Pape et al. . A serieVesicle formulations were prepared by slightly modifying the film hydration method . The surSize distribution of the DPPC/Surfactants vesicles was studied by optical microscopy using a Zeiss  light microscope equipped with a Linkam LTS120 hot stage, controlled by a PE94 unit. Images were acquired with a Canon PowerShot S90 Wide Zoom digital camera. Sizing was performed using ImageJ software of calibrated images .The binding mechanisms of surfactants inside the peptidoglycan glycosyltransferase protein were explored using Autodock Vina molecular docking simulation . The tarHOMO), lowest unoccupied molecular orbital energy (ELUMO), and gap energy between ELUMO and EHOMO (ΔE= EHOMO − ELUMO). Further, other molecular reactivity descriptors were calculated: electronegativity (χ) and chemical hardness (η) of the two molecules, which are given by the following equations [Quantum chemical methods and molecular modeling techniques help to define a large number of molecular descriptors characterizing the reactivity, form, and binding properties of a complete molecule as well as molecular fragments and substituents. The use of theoretical parameters can help to design a theoretical approach that explains the DPPC/surfactant interactions . Computaquations ,41.(2)χα-acyl-tryptophan and Nα-acyl-Phenylaniline-based surfactants  requires connecting the aliphatic chain to the amino acid. The synthesis was carried out in 2 steps: (a) N-acylation of the α-amino group of the amino acid with the corresponding fatty acid chloride, the reaction progress was followed through HPLC, and after 4 h, the conversion was higher than 90%, and (b) the second step involves the building of an amide bond by condensation of the methyl ester group of the CnPOM/CnTOM with one of the amine groups of the 1,3-diaminopropane, followed by acidification with HCl in a methanolic solution ; on the other hand, for the same head group, it was observed that the pKa decreased as the alkyl chain increased. It seems that with increasing alkyl chain lengths, the hydrophobic chains become more tightly packed; then, to avoid unfavorable repulsions of the positively charged polar heads, the surfactants release more protons, showing lower pKa values. This behavior agrees with that obtained for Nε-lysine based surfactants [On the one hand, for the same alkyl chain, similar pKfactants  and phenfactants .a of these surfactants is higher than that reported for amino-acid-based surfactants with the cationic charge located on the α-amine group [a of the protonated amine groups is greater when the amine group is separated from the other functional groups of the molecules by some CH2 groups.The pKne group  and agrene group . These ra of the C8-C12 derivatives are equal to or higher than 7.5. At pH equal to the pKa, the compound is 50% protonated, while for pH values 1 unit lower than the pKa, it can be considered that the pH-sensitive surfactants are totally protonated. Then, it can be stated that the C8-C12 derivatives are totally protonated at the pH used to measure the antimicrobial activity, while the C14 derivative maintains around 50% of its cationic charges. The obtained pKThe electrical conductivity of surfactant solutions changes to different extents below and above the CMC . The hyp1) and third (I3) vibrionic peaks of pyrene in the emission spectra is a polarity index for the microenvironment of the fluorescent probe. The CMC and the degree of ionization were determined. The values obtained are listed in 1/I3 ratio of the emission spectra plotted as a function of surfactant concentration. At the initial concentrations, the ratio was about 1.6, which is comparable to that of pyrene in pure water. At the most dilute concentrations, no significant changes in the I1/I3 ratio were observed; due to the absence of surfactant aggregates, the polarity of the microenvironment around the pyrene molecules is the same. Then, an abrupt decrease in I1/I3 intensity is observed, indicating the formation of aggregates and the incorporation of pyrene into the micellar core. The data were fitted to Boltzmann-type sigmoidal curves, and the CMC values were taken as the midpoint of the transitions. In general, the CMC values derived from the fluorescence measurements are of the same order as those determined by conductivity. The I1/I3 ratio above the CMC was similar for both families of surfactants, suggesting that their micelles had comparable polarity. Note that the preparation of C14PC3NH3Cl at high concentration is complicated by the increase in viscosity observed for these solutions.The figure shows the changes in the Iα-alkyl-arginine methyl ester  have a larger molecular volume than only one ring (in the case of phenylaniline), we would expect the phenylaniline homologs to have bigger CMC than the tryptophan derivatives. Our experimental results contradict this expectation, probably because of the presence of the indole. In a comparative study based on the theoretical calculations of AB initio, Steve Schreiner et al. […N bond involving histidine is the strongest, followed by the OH…O bond where tyrosine acts as a donor, and then by the NH…O bond of tryptophan. If such bonds are unattainable, or in the case of phenylalanine that contains no heteroatoms, other stabilizing interactions are possible, albeit somewhat weaker. These results suggest that the presence of the indole group in the tryptophan-based surfactants increases their hydrophilic character, which implies an increase in their CMC values compared to that of CnPC3NH3Cl derivatives.The CMC values of the two families studied, N-acyl-tryptophan salts and N-acyl-phenylalanine salts in water, depended on the alkyl chain length. For homologs with the same polar head, the CMC decreases with an increasing number of carbon atoms in the hydrophobic chain. In all cases, the CMC values are of the same order for the different polar headgroups, as expected. More hydrophilic species should be more soluble and, therefore, less prone to form micelles. These CMC values are significantly lower than those of surfactant-based amino acids with a linear hydrocarbon N= 5.8 mM ) and Nα-= 7.2 mM ). The inr et al.  showed t14PC3NH3Cl, C12PC3NH3Cl, C14TC3NH3Cl, and C12TC3NH3Cl) were chosen to formulate cationic vesicles based on DPPC. Four molar ratios were studied for the mixtures DPPC/Surfactants: 80:20, 60:40, 40:60, and 20:80. Vesicle morphology, shape, and size were monitored by optical microscopy. Phospholipid vesicles (liposomes) are under investigation as models for biological membranes and as carriers for various bioactive agents such as drugs, diagnostic and genetic materials, and vaccines . The fouAccording to these results, it can be stated that large vesicles are present in all vesicle formulations. There are not clear trends in the determined sizes; although the median diameters may be significantly different, the ranges of observed diameters largely overlap, blurring possible composition effects. With the observed sizes, the use of light scattering for the determination of sizes and for the determination of z-potential was not possible . 2)10CH3 or a -(CH2)12CH3 alkyl chain  with exq value of 0.1 Å−1. This scattering pattern is due to the electronic distribution present in phospholipid bilayers with a low electronic density region at the center of the bilayer (corresponding to the methyl and methylene groups of the hydrophobic chains) and two high electronic density regions corresponding to the location of the phosphatidyl-choline groups [q values . In 12TC3NH3Cl, together with the best fit of these data. The surfactant alone is also shown for comparison; however, this could not be sensibly fit to a bilayer model. In To further investigate if these surfactants can be incorporated into cationic vesicles, we mixed these compounds with DPPC. The samples were prepared to induce the formation of vesicles. DPPC produces a scattering pattern typical of vesicles see  with a we groups . This ca12TC3NH3Cl appears at a slightly smaller q than that of C14TC3NH3Cl. Some more different are the electronic density profiles. Comparing 12PC3NH3Cl and C14PNH3Cl. Both the graphs and parameters are shown in the From these results, we can conclude that the surfactants incorporate into the bilayers without producing dramatic changes in the structures of the bilayer up to high surfactant contents. All of the samples were unilamellar within the detection sensitivity, which can be estimated to be about 10%. Very similar results were found for CBacillus subtillis, Staphylococcus epidermidis, Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Escherichia coli, Acinetobacter baumannii, and Klebsiella aerogenes). The concentration tested for all surfactants ranged from 500 to 2 µM. The obtained MIC values (concentration required to completely inhibit the growth of microorganisms), as well as the MBC values (concentration of surfactants required to kill microorganisms), are summarized in The antibacterial activity was tested against eight representative bacteria strains   and dipeDifferent results have been reported regarding the effect of the aromatic group on antibacterial activity. It seems that the presence of aromatic rings enhances the antimicrobial effectiveness. For example, benzalkonium bromide, a compound with a benzyl group on the polar head, showed higher activity than its counterpart, dodecyl trimethyl ammonium ,61, and The MBC of these cationic surfactants, the concentration at which >99.9% of bacteria are killed, is also displayed in 12TC3NH3Cl. The MICs of this table correspond to the concentration of the cationic surfactant in the formulation. The antimicrobial activity of these systems depends on the ratio of DPPC/surfactant. The formulation with 80% of C12TC3NH3Cl exhibited similar antimicrobial activity to the pure surfactants. This means that these vesicles can act as carriers and antimicrobial agents simultaneously. The incorporation of higher percentages of DPPC in the formulations results in an important increase in the MIC values. In fact, formulations containing only 20% of this cationic surfactant did not show activity against any of the tested bacteria. This behavior was also observed for diacyl-glycerol arginine-based surfactants; the pure cationic surfactant exhibited good antimicrobial activity, while its vesicles had very low effectivity against bacteria [h = 79 nm) penetrated more deeply into the cell surfaces and, consequently, had lower MIC values than DODAB large vesicles (Dh = 500–800 nm) [bacteria . These rbacteria . The SAX–800 nm) . Cationi–800 nm) . It was –800 nm) . In this14TC3NH3Cl, DPPC/C12PC3NH3Cl, and DPPC/C12PC3NH3Cl are not very different from those shown by the DPPC/C12TC3NH3Cl, however, unexpectedly, these formulations did not exhibit effectivity against any of the tested bacteria. This behavior can be ascribed to the different affinity between the surfactant molecules with DPCC during vesicle formation  was calculated from plots of % hemolysis as a function of surfactant concentration , only about 30% of hemolysis was obtained. This means that this surfactant shows excellent selectivity against Gram-positive bacteria and A. baumani.The biomedical applications of cationic surfactants depend on their ability to selectively interact with bacterial membranes without toxic effects on human cells. The phenylalanine CThe major component of the bacterial cell wall is a cross-linked glycopeptide polymer called peptidoglycan. This polymer surrounds the cytoplasmic membrane of bacteria and functions as an exoskeleton, maintaining cell shape and stabilizing the membrane against fluctuations in osmotic pressure. A functioning peptidoglycan pathway is required for bacterial cell growth and division, and compounds that inhibit peptidoglycan biosynthesis have antibiotic activity .Molecular docking studies were carried out to understand the observed antimicrobial activities of the prepared surfactants and to shed light on the binding modes between docked ligands and enzyme targets ,70,71.The likeliest docked positions of surfactant ligands with the best binding affinity for ligands complexes in the active site of the targeted receptor are shown in All surfactants showed a good affinity to receptor pocket , with fr2, hydrophobic parts, and aromatic rings participate in the mode of interaction against different residues in the receptor pocket.The phenylalanine and tryptophan-based surfactants show three types of interactions: electrostatic, hydrogen bonding, and hydrophobic. The functional groups of the molecules, carbonyl, secondary amine NH12 to C14. Similar behavior has been observed for the C10 and C12 tryptophan derivatives, while the transition from C12TC3NH3Cl to C14TC3NH3Cl leads to a decrease in the number of hydrophobic bonds from 2 to 1.The elongation of the hydrophobic alkyl chain does not affect the number of hydrophobic interactions. For the phenylalanine derivatives, the hydrophobic interactions move from 2 to 3 when the alkyl chain increases from C10TC3NH3Cl, C14TC3NH3Cl, and C10PC3NH3Cl, which present attractive charge interactions in their mode of action. It is not easy to justify a single effect or a combination of effects that can fully explain the differences in reactivity of the ligand in the protein pocket. The attractive–charge interaction distances observed in the mode of action of some ligands can be attributed to the existence of intramolecular interaction Charge-Dipole type between the N+ positive charge on the atom and the lone pair of the oxygen atoms attached to the carbonyl’s functions. This can also be explained by the degree of steric hindrance which exists at the level of the polar heads of the surfactants, which will prevent the contribution of N+ in the electrostatic interaction with the amino acid residues.All inhibitors show different numbers of interactions, except the CHOMO), the energy of the low unoccupied molecular orbital (ELUMO), energy gap (ΔE), hardness (ɳ), and electronegativity (χ). These parameters can help to correlate the electronic distribution of the DPPC/Surfactants vesicles and their antibacterial activity. The parameter values are shown in Several electronic parameters in terms of chemical descriptors were calculated based on the geometrical optimization of the new surfactants molecules: energy of the highest occupied molecular orbital (E12 and C14 derivatives. The EHOMO (higher energy) is often associated with the ability of the molecule to donate the electron, and the ELUMO (lowest energy) indicates the higher probability that the molecule would accept electrons [HOMO of these surfactants is less negative (−6.1851eV to −6.5943 eV) than that of the DPPC (−7.4133 eV). This indicates that surfactant molecules are more likely to give electron density than DPPC molecules. Then, it is expected that when the pairs of DPPC/Surfactants interact, the tendency of electronic charge transfer will be performed easily from the surfactant’s molecules to the DPPC.All these parameters were assessed to elucidate the DPPC interaction with the Clectrons . It can LUMO − EHOMO reflects the polarizability and the reactivity of a molecule [The gap energy ΔE = Emolecule ,74. The molecule . The invThe Hard–Soft–Acid–Base theory (HSAB) classifies reactive species as relatively “hard” or “soft” based on polarizability, i.e., the ease with which electronic density can be shifted or delocalized ; hard moFrom the calculated chemical descriptors, it can be assumed that when DPPC interacts with those surfactants, DPPC molecules play the role of electron density acceptor while surfactant molecules act as donors.int) according to D.C. Young [DPPC is the total energy of the DPPC molecule, ESurfactant is the total energy of the surfactants compounds, and EDPPC_Surfactant represents the total energy of the new complex system formed between the surfactants and the DPPC.The loss of the biological activity of these liposomes can be attributed to the strong molecular affinity between DPPC and surfactants. This interaction affinity between DPPC and surfactant molecules can be determined by calculating interaction energies (noted EC. Young  by using12TC3NH3Cl is higher than that of the other three complexes, which reflects a lower affinity between DPPC and C12TC3NH3Cl. Due to the low affinity between C12TC3NH3Cl and DPPC, the cationic surfactants in these formulations will be more available to interact with the bacterial walls and, consequently, maintain some antibacterial activity. However, for the C12PC3NH3Cl, C14PC3NH3Cl, and C14TC3NH3Cl, the strong affinity between the surfactant molecules with DPCC in the vesicle will reduce the availability of surfactant molecules to be active against bacteria. Shao et al. found that the strong interactions between phospholipids vesicles and ionic-charged chitosan/alginate multilayers give rise to perfect fluid lipid bilayers on polyelectrolytes multilayers. Coarse-grained molecular dynamics simulations suggested that the formation of these fluid bilayers follows the parachute model [All complexes formed between DPPC and surfactants present negative energies of interaction , indicatte model . A similCationic amino-acid based-surfactants have been successfully prepared using tryptophan and phenylalanine. The synthetic approach used to prepare these compounds fits some of the green chemistry requirements: utilization of renewable starting materials and a synthetic procedure consisting of two simple chemical reactions that do not require the use of an organic solvent or protecting amino acids.a decreases as the hydrophobic chain length increases, with phenylalanine being consistently more acidic than tryptophan. The CMC of the surfactant was determined primarily by the hydrophobic/hydrophilic balance. It was found that the presence of the indole group in the structure of tryptophan surfactants increases the hydrophilic character and, consequently, the CMC of the CnTC3NH3Cl homologs. The micelles of both families of surfactants based on dodecyl and tetradecyl hydrophobic chains can be fitted to cylindrical models, with C14PC3NH3Cl producing the most elongated micelles, agreeing with the macroscopically observed viscosity of their solutions.Both headgroups can be considered weak acids, where apparent pK12-C14 alkyl chains exhibited good antimicrobial activity against Gram-positive and Gram-negative bacteria, and the type of amino acid did not affect their antimicrobial activity. Vesicles containing 60 and 80% of C12TC3NH3Cl exhibited good antimicrobial activity; the effectiveness against these microorganisms decreased drastically when the percentage of DPPC increased. However, vesicles containing phenylalanine derivatives and the C14 tryptophan homolog did not show activity at the tested concentrations. DFT simulation suggests that the lack of activity observed for these vesicles could be ascribed to the high affinity between DPPC and these surfactants.SAXS investigations show the formation of unilamellar vesicles for all DPPC/surfactant ratios; moreover, only the bilayer thickness of vesicles with higher surfactant contents was significantly affected. Surfactants with CMolecular docking results obtained using the enzyme peptidoglycan glycosyltransferase suggest the existence of three types of interactions that could be the basis of some of the antimicrobial mechanisms.


Background: Russia’s aggression against Ukraine in early 2022 resulted in a large migration of refugees to many countries, including Poland. Vaccination coverage for some infectious diseases in Ukraine is lower than in Poland; consequently, the incidence of infectious diseases—including measles, poliomyelitis, tuberculosis, and COVID-19—is higher. We aimed to investigate whether the migration of Ukrainians had influenced decisions of Polish parents on having their children vaccinated and to examine their attitudes towards vaccinations. Material and methods: A cross-sectional online survey study was designed. Data on parents’ demographics, attitudes toward vaccination, and knowledge of the current epidemiological situation in Poland were collected. Parents participating in the study were divided into two subgroups for further analysis according to their decisions to have their children vaccinated. Results: A total of 568 questionnaires were collected, of which 21 did not meet the inclusion criteria for the analysis (respondents were not parents). The Ukrainian immigrants’ influx affected 54 (9.87%) participants in their decision of having their children vaccinated. Respondents in this group were more likely to have a positive attitude toward recommended vaccinations (p = 0.0428); in addition, they more often had their children vaccinated with recommended vaccinations (p = 0.0063), believed the vaccination coverage with mandatory vaccinations was higher in Poland than in Ukraine (p = 0.0014), and believed the incidence of diseases covered by mandatory (p = 0.0472) and recommended (p = 0.0097) vaccinations was higher in Ukraine. In addition, parents who declared that the migration had affected their decision regarding their children’s vaccinations had more often been vaccinated due to the influx of Ukrainian immigrants (p < 0.00001) and were more likely to be aware of how migration had impacted the current epidemiological situation in Poland (p = 0.0021). Moreover, the survey more often made these participants think about getting additional vaccinations for themselves (p < 0.0001) and their children (p < 0.0001). Conclusions: The Ukrainian immigrants’ influx affected nearly one tenth of surveyed parents in their decision of having their children vaccinated. This group was more aware of the differences between infectious diseases’ epidemiology in Poland and Ukraine. In addition, they also had a more positive attitude toward vaccinations. Hemophilus Influenzae type B (Hib). Hepatitis B, meningococcus, pneumococcus, chickenpox, influenza, and tuberculosis should also be considered depending on the season, epidemiological situation, and living conditions [Russia’s aggression against Ukraine in February 2022 resulted in a large migration of refugees to many countries. As of January 2023, an estimated 9.5 million people have crossed the Polish border since the war began . Among tA cross-sectional survey study was designed. The questionnaire included 25 single-choice and 4 multiple-choice questions on demographics , whether respondents were parents of a child/children under 18, their attitudes toward vaccination, and their knowledge of the current epidemiological situation in Poland. The division into mandatory and recommended vaccinations was carried out on the basis of the law regulating the program of immunization in Poland in 2023.Data were collected through an online survey, which was created using the “Google Forms” application between 15 November 2022 and 4 January 2023 and distributed via social media (Facebook and Instagram) on forums and social media groups/accounts related to parenting issues. Each participant was asked to complete the survey only once. There was no time limit to access the form. After completing the survey, participants received feedback on the epidemiological situation in Ukraine and Poland for education purposes.After collecting responses, the study group was divided into two subgroups for further analysis. Stratification was performed according to the declared impact of Ukrainian migration on participants’ decisions as to whether they would vaccinate their children or not.Inclusion criteria were completing the form and being a parent of at least one child under the age of 18.By completing the questionnaire, each respondent agreed to anonymously participate in the study and gave consent for publication of the results of this study. The exclusion criterion considered the indication that one is not a parent of a child/children under 18 years of age. Twenty-one surveys were rejected because respondents answered that they were not parents.2 or Fisher exact test were used to evaluate categorical variables. A p-value of <0.05 was considered significant. Statistical analysis was performed using Quick Statistics Calculators .The Mann–Whitney U test was performed to compare continuous variables and the chiIn total, 568 questionnaires were collected, of which 21 were excluded due to not meeting the inclusion criteria. The median age of the study participants was 34 (interquartile range (IQR): 31–39 years). The majority were females (503/547 (91.96%). Cities with over 500,000 inhabitants were the place of residence for 179/547 (32.72%) respondents, and a college degree was obtained by 472/547 (82.29%).A positive attitude toward vaccination was declared by 497/547 (90.86%) study participants. According to the surveyed population, the Ukrainian immigrants’ influx had affected 54/547 (9.87%) people in their decision of having their children vaccinated. No influence on this decision was declared by 493/547 (90.12%) participants.p = 0.0428), have their children vaccinated with recommended vaccinations , were more likely to believe the vaccination rate with mandatory vaccinations was higher in Poland than in Ukraine , believed that the incidence rate of diseases covered by mandatory vaccinations was higher in Ukraine than in Poland , believed the incidence rate of diseases covered by recommended vaccinations was higher in Ukraine than in Poland , were more likely to have been vaccinated due to the influx of Ukrainian immigrants , and were more likely to be aware of how the influx of migrants from Ukraine had affected the epidemiological situation in Poland . More often than not, the survey made participants whose vaccination decisions were influenced by the migration of Ukrainians think about getting additional vaccinations  and having their children vaccinated with additional vaccines   See .There was no significant difference in vaccines chosen by parents between the two groups. Participants most often chose for themselves to be vaccinated against pertussis , SARS-CoV-2 , and influenza , while they intended to be vaccinated against influenza , SARS-CoV-2 , meningococcal disease , and pertussis . In an analogous question about the participants’ children, most children had already been vaccinated against meningococcal disease , influenza , and varicella . Likewise, their parents most often intended to have them vaccinated against varicella , meningococcal disease , and influenza (13.2.38%).p = 0.9891).The problem of evading mandatory vaccinations is growing worldwide, including Poland. It poses a huge threat to the public health in this country. In 2015, in Poland, infectious diseases caused about 1900 deaths, of which 300 deaths were due to hepatitis B and more than 500 due to pulmonary tuberculosis. Both diseases may be effectively prevented with mandatory vaccinations implemented within the first year of life . Betweenp = 0.0063).The following Polish studies examined parents’ feelings about recommended vaccines: according to Janosz J. et al., a positive attitude towards the recommended vaccinations was recorded in 60% of the parents who had children under 3 years of age ; accordip = 0.03). The study also showed that working parents were more likely to report willingness to have their children vaccinated compared with unemployed parents . In contrast, no differences were found in terms of age, place of residence, education level, marital status, number of children in the family, or religiosity [p = 0.002) or education level (p = 0.004). Among respondents using recommended vaccinations, those aged 18–30 years and those with tertiary education had the highest proportion [p = 0. 0428).Male parents, parents who had a university education, and parents who had not been exposed to adverse reactions after vaccination in the past were more likely to report that their children have received the recommended vaccinations . Moreoveigiosity . Dąbek Joportion . In our p = 0.0021) and were more likely to have been vaccinated due to these circumstances .The influx of many refugees from Ukraine may pose a potential epidemiological threat. It is associated with infectious diseases including, e.g., COVID-19, measles, and poliomyelitis . Rzymskip = 0. 0014), believed that the incidence rate of diseases covered by mandatory vaccinations was higher in Ukraine than in Poland . This indicates that they were more aware of the current epidemiological situation regarding infectious diseases in both countries.Lack of awareness regarding the need for vaccination is one of the main factors contributing to vaccine hesitancy and avoiding mandatory vaccines . Greaterp = 0.0097). There was no significant difference between the two subgroups regarding awareness of the vaccination rate with recommended vaccinations, vaccination rate with SARS-CoV-2 vaccine, and COVID-19 incidence in Poland and Ukraine.According to the survey study conducted by Zaprutko T. et al., Poles were significantly more likely to be vaccinated against influenza and COVID-19 than Ukrainians. In addition, Ukrainians were less likely to oppose making these vaccinations mandatory . In our p < 0.001) and having their children vaccinated with an additional vaccine .According to Jarret C. et al., dealing with vaccine hesitancy includes increasing vaccination knowledge and awareness . In a stThis study has limitations. The questionnaire did not contain restrictions that prevented one person from completing it more than once. The survey was conducted more than six months after the beginning of the influx of migrants from Ukraine. Over time, respondents may have become accustomed to the situation by paying less attention to it. In addition, data were collected through an online survey. The opinions of parents who were very busy and had less access to the internet might have been excluded from this study. However, there are also a few strengths worth mentioning. A large population of people was surveyed and the questions were straightforward and easy to fill . In addition, the questionnaire was completely anonymous; therefore, study participants were encouraged and willing to give honest answers.The Ukrainian immigrants’ influx affected 9.87% of surveyed parents in their decision to have their children vaccinated. This group was more aware of the differences in infectious diseases’ epidemiologies in Poland and Ukraine. In addition, they had a more positive attitude toward vaccinations and, more often, the survey made them think about getting additional vaccinations for themselves and their children.


Birth weight is a crucial factor linked to a newborn’s survival and can also affect their future health, growth, and development. Earlier, researchers focused on exploring maternal and fetal factors contributing to low birth weight. However, in recent years, there has been a shift toward effectively predicting low birth weight by utilizing a combination of variables. This study aims to develop and validate a nomogram for predicting low birth weight in Ethiopia.A retrospective follow-up study was conducted, and a total of 1,120 pregnant women were included. Client charts were selected using a simple random sampling technique. Data were extracted using a structured checklist prepared on the KoboToolbox  and exported to STATA version 14  and R version 4.2.2  for data management and analysis. A nomogram was developed based on a binary logistic model, and its performance was assessed by discrimination power and calibration. Internal validation was performed using bootstrapping. To evaluate the clinical impact, decision curve analysis was applied.The nomogram included gestational age, hemoglobin, primigravida, unplanned pregnancy, and preeclampsia. The AUROC of the predicted nomogram was 84.3%, and internal validation was 80.1%. The calibration plot indicated that the nomogram was well calibrated. The model was found to have clinical benefit.The nomogram demonstrates strong discrimination performance and can predict low birth weight clinically. As a result, it can be used in clinical practice, which will help clinicians in making quick and personalized predictions simply and rapidly, enabling the early identification and medical intervention. For broader applicability, the nomogram must be externally validated. Birth weight is a reliable indicator of fetal development and might have a significant role in predicting the newborn’s survival and can also affect their long-term health, growth, and development . Low birGlobally, 60–80% of neonatal mortality is caused by LBW . Infant Maternal age, birth order, household income, primigravida, educational level, rural residence, diet, anemia, parity, presence of chronic illness, maternal nutritional status before and throughout pregnancy, preeclampsia, short stature, sex of the newborn, physical labor during pregnancy, and antenatal care (ANC) are well recognized risk factors for LBW , 15, 17.The most effective strategy for preventing LBW is to initiate prenatal care as early as possible in pregnancy and to continue receiving it throughout pregnancy . By the A retrospective follow-up study was conducted at Debre Markos Comprehensive and Specialized Hospital (DMCSH) in Ethiopia to develop and validate a nomogram for LBW among pregnant women who had antenatal care visits.t” is a function of various prognostic determinants measured at a time point before the occurrence of LBW or during pregnancy, “t0.” The study’s domain included pregnant women who received antenatal care.The study’s hypothetical design was proposed as follows: the incidence of LBW at a future time “The study was conducted at DMCSH from 1 January 2020 to 30 August 2022, and the data were extracted from 3 January 2023 to 1 February 2023. DMCSH is located at Debre Markos town, which is 299 km from Addis Ababa, Ethiopia’s capital city and 265 km from Bahir-Dar, the capital city of the Amhara Regional State. This hospital is one of the largest tertiary-level referral facilities in the Amhara region. The history of this hospital dates back to 1965 when it was founded by Emperor H/Selassie, and currently, it serves approximately five million people . GynecolN = (n*10)/I, where n represents the number of predictors (N = (n*10)/I, (15*10)/0.140 = 1,071. Accounting for a 5% for missing data, the adjusted sample size was 1,124. Using a computer-generated simple random sampling procedure, 1,124 records (charts) of study participants were selected.A rule of thumb for developing a prediction model suggests having 10 to 20 events for each predictor . Generaledictors . In thisedictors , and theLow birth weight (yes/no).Age, marital status, middle upper arm circumference (MUAC), maternal weight, place of residence, gravidity, hemoglobin level, pregnancy status, timing of ANC initiation, timing of iron folate initiation, comorbidities (such as pulmonary: history of asthma or COPD (chronic obstructive pulmonary disease); cardiac: history of heart failure or ischemic heart disease; and renal diseases), and preeclampsia.Mothers with hemoglobin level less than 11 g/dL . Within 72 h of delivery, the birth weight was measured on a digital scale  guideline was used for developing and reporting the prediction model . Tables p-value of ≤0.25 in the bivariable analysis were fitted to the multivariable regression analysis. After a stepwise backward elimination technique was used, the role of each predictor in the multivariable analysis was assessed by the likelihood ratio test. To be more liberal, a p-value ≤0.15 for the likelihood ratio test was used to fit the reduced model  was used to evaluate the discrimination power of nomogram. The AUROC between 0.7 and 0.9 indicates good discrimination power and > 0.9 indicates excellent discrimination power , 24. ModWe included 1,120 women who gave birth and had their birth weight taken within 72 h of delivery. The demographic, obstetric, and clinical features of pregnant women enrolled in the study are shown in p-value ≤0.25 in the univariable analysis. Using the results, a prediction model was developed, and the equation for the prediction model was obtained  had LBW newborns (SD: 516.1). The average birth weight was 2,942.7 g. Following a study of the literature, 14 baseline measurements on the mother’s demographic, obstetric, and clinical features were taken into consideration to predict LBW. Numerous factors were determined to be eligible for inclusion in the prediction model using the univariable analysis. Age at current pregnancy, hemoglobin level, gravidity, marital status, residency, weight, MUAC, timing of ANC initiation, pregnancy status, and preeclampsia were the variables with a obtained .Probability of LBW = f (predictor variables).PR (LBW) = f .PR (Y = 1) = f (X), where Y = 1 refers to having LBW and Y = 0 refers to not having LBW.The AUROC of the nomogram using five predictors was 84.3%  using original beta coefficients, which means a model that was 84.3% differentiates pregnant women who give birth to LBW from those who do not give birth to LBW, indicating the model’s capacity for identification was good .p = 0.729) and the calibration plot fell along a 45° line . it indicates that the model well represented the data .Since the model was well calibrated , with the equivalent total points in the nomogram showing a low risk of the LBW. For a pregnant women aged <20 and having preeclampsia, the total point was as follow: 7.4 + 7.6 = 15.0 points. Thus, the probability of giving birth to an LBW baby, which is approximately 0.86 (86%), is high.The best cutoff point value (Youden index) to predict a high or low risk probability of LBW was 0.220 (22.0%). The Youden index cut point value has a maximum sensitivity and specificity of 78.0 and 91.0%, respectively. At the Youden index threshold value, the positive and negative predictive values were 79.4 and 86.1%, respectively. The AUROC curve at the optimal cut point was 80.0%.For internal validation of the nomogram, 1,000 random bootstrap samples with replacement were generated from the dataset that included complete data on all predictors. After bootstrapping, the predictive performance was 0.801(80.1%) , which iIn this study, we constructed a nomogram model based on the optimal combination of maternal characteristics to predict the risk of LBW during pregnancy in areas where no laboratory tests and ultrasound scans are not included. This model includes factors such as age under 20 years, hemoglobin levels below 11 mg/dL, primigravida, unplanned pregnancy, and preeclampsia.The nomogram approach provides a visual and individualized prediction, which can make it easier for obstetricians to evaluate each pregnant woman’s risk of LBW based on her score and subsequently provide individualized treatment. Furthermore, the nomogram model makes it simple and straightforward for pregnant women who are personally at risk for LBW babies to participate in the intervention and receive superior clinical results. Additionally, this study’s predictive model was developed using the maternal clinical data from early pregnancy. It can be used for screening expectant mothers for LBW at this early stage of pregnancy, allowing for the earlier implementation of effective intervention and treatment for these pregnant women. More significantly, the screening tool’s variables are simple to understand.Several approaches were used to assess the nomogram’s performance and discrimination. The AUROC value of 0.843 proved that the nomogram had good discrimination power . SimilarThis study conducted for predicting LBW in India revealed an AUC value of 0.762 . SimilarThe developed model was well calibrated, showing its reliability , and it revealed similar discrimination performance after internal validation, indicating that it is capable of predicting LBW in an independent set of pregnant women with a comparable degree of accuracy. The decision curve analysis showed that the established model for early risk classification of pregnant women for LBW has a higher clinical benefit than treating none and treating all pregnant women across a wide range of threshold probability. The concept of decision curve analysis involves the standard net benefit along the threshold probability, which is determined by the difference between benefit and cost. In this study, the benefit refers to treating true positives (after correctly predicting a pregnant woman will have an LBW baby) while the cost is treating false positives (after incorrectly predicting a pregnant woman will have an LBW baby).First, the study was conducted with a sufficient number of participant outcomes for LBW prediction. This helped in building the model that has an adequate number of predictor variables while preventing overfitting. Second, we have developed a visual diagnostic tool for LBW that enables patients and physicians to make individualized predictions quickly and easily. Additionally, local governments and healthcare providers can use our prediction tool as a guide to enhance pregnancy outcomes.The study was not without limitations; it would have been better if it had been conducted using a prospective follow-up study design. In retrospectively collected data, some variables that predict LBW might have been missed. However, the nomogram developed using retrospectively collected data are still important in resource-limited settings, including Ethiopia. Additionally, the model was not externally validated using an independent dataset. It would have been better if it had gone through external validation to ensure its prediction capability when applied to other contexts.In conclusion, this study developed and validated a clinical nomogram for the prediction of LBW risk in Ethiopia. In areas with no laboratory tests and ultrasound scans, a woman’s maternal history can be used to determine the risk of a LBW baby. Factors such as being under the age of 20, having hemoglobin levels less than 11 mg/dL, being a primigravida, having an unplanned pregnancy, and experiencing preeclampsia are all independent risk predictors of LBW. We found that the discrimination ability of this nomogram is highly valuable and can provide significant clinical and public health benefits. We recommend clinicians to utilize this nomogram using the appropriate cutoff point provided to categorize pregnant women. For researchers validating the prediction tool in another context, this would also be a useful approach.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving humans were approved by Institutional Review Boards (IRB) of the University of Gondar. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants' legal guardians/next of kin because We used secondary data. We only extracted clinically relevant information, did not affect the treatment and health of patients, and the patients' privacy was protected, so written informed consent was not applicable for this study.BF: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. GT: Data curation, Formal analysis, Software, Supervision, Validation, Writing – review & editing. TG: Supervision, Validation, Visualization, Writing – review & editing. MA: Software, Software, Validation, Visualization, Writing – review & editing.


Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental condition typified by inattention, hyperactivity, and impulsivity. Comorbid psychiatric disorders are common among children and adolescents with ADHD. In this study, it was aimed to examine anxiety and somatic symptoms in children and adolescents with ADHD and the effect of methylphenidate treatment on these symptoms.  Three groups were formed, consisting of 37 children and adolescents diagnosed with ADHD and received methylphenidate treatment, 37 newly diagnosed, treatment-naive children and adolescents with ADHD diagnosis, and 37 children and adolescents without the diagnosis of ADHD. The symptoms of ADHD in children were examined by using the DSM-IV-based child and adolescent behavior disorders screening and rating scale, the symptoms of anxiety were examined by using the screen for child anxiety-related disorders (SCARED), and somatic symptoms were examined by using the DSM-5 level 2 somatic symptom scale.  In the newly diagnosed, treatment-naive with ADHD group, anxiety and somatic symptoms were found to be significantly higher compared to the ADHD group with methylphenidate treatment and the non-ADHD group. It was shown that the symptoms of panic disorder, generalized anxiety, and social phobia were observed more in the newly diagnosed, treatment-naive with ADHD group compared to the treatment group with ADHD.  It was determined that children and adolescents diagnosed with ADHD had more anxiety and somatic symptoms. Anxiety and somatic symptoms increased as the severity of ADHD symptoms increased. Anxiety and somatic symptoms were lower than in ADHD children receiving methylphenidate treatment. Clinicians should keep in mind to evaluate anxiety and somatic symptoms in children and adolescents with ADHD before the treatment. Attention-deficit hyperactivity disorder (ADHD) is identified as a neurodevelopmental disorder typified by symptoms of inattentiveness, hyperactivity, and impulsiveness. These symptoms negatively affect the social, school, and work-related activities . The preComorbid psychiatric disorders accompany 40-80% of children with ADHD . JehnsenThe incidence of anxiety disorder is 6.5% in children and adolescents . The coeSomatic symptoms are common in children and adolescents . It was Psychostimulants are the first choice in the treatment of ADHD . StudiesDue to children and adolescents with AHDH diagnosis having anxiety and somatic symptoms, and to our knowledge, there are limited studies on this issue, it was aimed to examine the frequency and dispartion of anxiety and somatic symptoms in children and adolescents with ADHD. It was also aimed to investigate the methylphenidate treatment effect on anxiety and somatic symptoms in children and adolescents with ADHD.A total of 111 children and adolescents, including 37 newly diagnosed, treatment-naive children and adolescents with ADHD between the ages of 6 and 15, 37 children and adolescents diagnosed with ADHD receiving only methylphenidate (extended-release or immediate-release formulations) treatment for at least six months, and 37 children and adolescents without the diagnosis of ADHD as the control group, participated in the current study. Newly diagnosed, treatment-naive children with ADHD did not use any psychotropic medication. A psychiatric interview based on DSM-5 was held by a child and adolescent psychiatrist with all participants. All participants' parents filled out the DSM-IV-based child and adolescent behavior disorders screening and rating scale in order to assist the diagnosis of ADHD. The sociodemographic data form was given to parents to obtain the sociodemographic data. The parents filled out the screen for child anxiety and related disorder- (SCARED-) parent form to examine the symptoms of anxiety and the DSM-5 level 2 somatic symptom scale-parent form to examine somatic symptoms. Those with a score of 25 and above in the SCARED were excluded. Somatic symptoms were also evaluated by clinician in all participants. The patients who had an IQ score of less than 80, known neurological, genetic, and chronic medical diseases, and used medication related to it were not included. Also, having mental retardation, bipolar disorder, and psychosis were determined as exclusion criteria in all groups. The study was approved by the Trakya University Local Ethics Committee (TÜTF-BAEK 2019/400). All participants were informed about the current study, and their written consent was obtained.It is a 41-item scale developed by converting the DSM-IV diagnostic criteria into a question form. The scale has 9 items examining attention deficit, 6 items examining hyperactivity, 3 items examining impulsivity, 8 items examining ODD, and 15 items examining CD. To confirm the diagnosis of ADHD, at least 6 of 9 items questioning attention deficit should be rated as 2 or 3, or at least 6 of 9 items questioning hyperactivity and impulsivity should be rated as 2 or 3. The Turkish validity and reliability study of this scale has been carried out by Ercan et al. .It consists of 41 items and 5 subscales . It is accepted that a score of 25 and above in the SCARED is a warning for anxiety disorder. Its Turkish validity and reliability study has been carried out by Çakmakçı et al. .The parent form for children aged 6-17 is a Likert-type scale that evaluates the somatic symptoms in the last seven days. The total score is between 0 and 26, and higher scores indicate more severe somatic symptoms. Its Turkish validity and reliability study has been carried out by Sapmaz et al. .U and Kruskal-Wallis tests were used for the analysis of quantitative independent data. For analyzing qualitative independent data, the chi-square test was used. The relationship between the variables was analyzed using the Spearman correlation analysis. IBM SPSS 27.0 was used. The limit of significance was chosen as p < 0.05 for all statistics.Mean, median, minimum and maximum values, standard deviation, and percentage were used in descriptive statistics. The Mann–Whitney p > 0.05). Maternal educational level was lower in the newly diagnosed, treatment-naive with ADHD group (p > 0.05). There was no significant difference between the groups in terms of paternal educational level (p > 0.05). The distribution of the income levels of the families was similar in all groups (p > 0.05)  .p < 0.05). The total score was significantly higher in the newly diagnosed, treatment-naive with ADHD group compared to the treatment group with ADHD (p < 0.05)  .p < 0.05). When subscale scores were compared, the scores for panic/somatic symptoms, generalized anxiety, and school phobia were demonstrated to be higher in the newly diagnosed, treatment-naive with ADHD group compared to the treatment group with ADHD (p < 0.05). No significant difference was indicated in the panic/somatic symptoms, separation anxiety, generalized anxiety, and school phobia subscale scores between the treatment group with ADHD and the non-ADHD group (p > 0.05)  .The total score of SCARED is found to be significantly higher in boys than girls in the newly diagnosed, treatment-naïve with ADHD group. No significant difference was found between girls and boys in the total score of SCARED in the treatment group with ADHD and the non-ADHD group.p < 0.05). No significant difference was indicated between the ADHD group with treatment and the non-ADHD group (p > 0.05)  .No significant difference was found between girls and boys in the total score of the DSM-5 level 2 somatic symptoms scale in all groups.p = 0.002). A significant correlation was found between somatic symptom scale total scores and the SCARED total score (p = 0.001)  .It was observed that the children and adolescents with ADHD had more anxiety and somatic symptoms compared to the non-ADHD children and adolescents in this study which is compatible to the literature. Both anxiety and somatic symptoms were significantly higher in the newly diagnosed, treatment-naive with ADHD group compared to the children and adolescents diagnosed with ADHD receiving methylphenidate treatment. It was observed that anxiety and somatic symptoms increased as the severity of ADHD symptoms increased. In a study conducted with 102 children with ADHD, it was shown that while externalizing symptoms were mostly observed in hyperactive-impulsive type ADHD, anxiety, depression, delinquent behavior, and internalizing symptoms were mostly observed in the combined type. Somatic complaints were more common in boys .The total score of SCARED was significantly higher in the newly diagnosed, treatment-naive with ADHD group compared to the treatment group with ADHD and the non-ADHD group in this study. The symptoms of generalized anxiety, panic disorder, social phobia, separation anxiety, and school phobia were shown to be higher in the newly diagnosed, treatment-naive with ADHD group compared to the non-ADHD group. It was shown that the symptoms of panic disorder, generalized anxiety, and social phobia were observed more in the newly diagnosed, drug-naive ADHD group compared to the ADHD group with treatment. The ADHD group with treatment had more social phobia symptoms compared to the non-ADHD group. Souza et al. demonstrated that 23.5% of the children with ADHD were accompanied by anxiety disorders, and the generalized anxiety disorder (GAD) is the most common accompanying anxiety disorder by 12.8%, followed by social phobia by 3.84%, and separation anxiety by 3.8% in their study . In anotIt was shown that anxiety symptoms increased as the severity of ADHD symptoms increased in our study. Taurines et al. showed that the coexistence of ADHD diagnosis and anxiety disorders is associated with more attention problems, school phobia, mood disorder, and lower social ability compared to anxiety alone or ADHD alone . Most ofIn the current study, somatic complaints were observed to be significantly higher in the newly diagnosed, treatment-naïve children and adolescents with ADHD. Somatic complaints were observed to be less in the ADHD treatment group compared to the newly diagnosed, treatment-naive with ADHD group in the current study. Ahmann et al. demonstrated that irritability, staring, daydreaming, nail-biting, and anxiety were reduced by short-acting methylphenidate treatment. In the same study, appetite disturbance, stomachache, insomnia, headache, and dizziness increased with methylphenidate . BarkleyThere is no significant difference between girls and boys in terms of somatic symptoms in this study. In a study, it was reported that generalized anxiety, phobia, and being a girl were risk factors for somatic symptoms in children with ADHD, and children with ADHD and somatic symptoms had more anxiety symptoms . Also, cThe small sampling is the first limitation of this study for affecting the generability of the results. More studies conducted with larger sampling are needed. The data were obtained only from the parents. The data could be obtained from children and their teachers to increase the data diversity, so this is the second limitation. Making comparisons between three groups is the strength of the study. In conclusion, it was shown that children and adolescents with ADHD had more anxiety and somatic symptoms than children and adolescents without ADHD, and children and adolescents with newly diagnosed, treatment-naive with ADHD had more anxiety and somatic symptoms in this study. Less anxiety and somatic symptoms in children with ADHD who receive methylphenidate treatment may show the protective effect of ADHD treatment from comorbid conditions. Although it is known that somatic side effects are observed with psychostimulant treatment, the clinician should keep in mind that somatic symptoms may also be present before the initiation of the treatment.Based on the findings of current research, clinicians should evaluate and consider anxiety and somatic symptoms in children and adolescents with ADHD while planning and monitoring methylphenidate treatment. Future studies with larger sampling are needed to investigate anxiety and somatic symptoms in children and adolescents with ADHD and the effect of methylphenidate treatment on these symptoms due to mentioned limitations of this study.


Stimulation of the peripheral visual field has been previously reported as beneficial for cognitive performance in ADHD. This study assesses the safety and efficacy of a novel intervention involving peripheral visual stimuli in managing attention deficit hyperactivity disorder (ADHD).One hundred and eight adults, 18–40 years old, with ADHD, were enrolled in a two-month open-label study. The intervention  consisted of standard eyeglasses with personalized peripheral visual stimuli embedded on the lenses. Participants were assessed at baseline and at the end of the study with self-report measures of ADHD symptoms , and executive functions . A computerized test of continuous performance  was tested at baseline with standard eyeglasses and at the end of study using Neuro-glasses. The Clinical Global Impression-Improvement scale (CGI-I) was assessed at the intervention endpoint. Safety was monitored by documentation of adverse events.p = 0.037) and metacognitive functions concerning self-management and performance monitoring . A continuous-performance test (CPT-3) indicated significant improvement in detectability  and reduced commission errors (p = 0.004), suggesting that the Neuro-glasses have positive effects on response inhibition. Sixty-two percent of the participants met the response criteria assessed by a clinician (CGI-I). No major adverse events were reported.The efficacy analysis included 97 participants. Significant improvements were demonstrated in self-reported measures of inattentive symptoms (ASRS inattentive index; Neuro-glasses may offer a safe and effective approach to managing adult ADHD. Results encourage future controlled efficacy studies to confirm current findings in adults and possibly children with ADHD.Clinical trial registration: https://www.clinicaltrials.gov/, Identifier NCT05777785. Attention deficit hyperactivity disorder (ADHD) poses a significant public health problem estimated to affect up to 11% of children worldwide . SymptomAlthough ADHD has a wide range of adverse outcomes, it is one of the most treatable neurobehavioral disorders. Stimulants, such as amphetamines and methylphenidates, are a common ADHD treatment with documented safety and efficacy , 6. NeveIn this study, we use novel glasses, i.e., Neuro-glasses, as an intervention for treating adults with ADHD. It is worth noting that ADHD has been found to be associated with various visual problems and oculomotor abnormalities . A recenPeripheral vision provides essential information concerning our surroundings. It has a survival function as it guides and relates us to the world around us, showing high sensitivity to movement and brightness changes in the visual scene , 14. AltGiven the key role of peripheral vision in monitoring the environment and facilitating rapid detection of changes in the visual scene, it is expected to affect the allocation of attentional resources. Peripheral vision allows us to react quickly and shift attention to adjust our visually guided behavior to the visual scene. Without sufficient peripheral vision providing the visual context in which objects exist, it is difficult to distinguish relevant from nonrelevant information , 17–19. Previous studies have shown that peripheral stimuli external to a task often influence performance , 19–21. The beneficial effect of glasses using peripheral visual stimulation was previously reported in patients with chronic dizziness . It was Eligible participants included adults aged 18 to 40 with a documented history of ADHD diagnosis. The upper age limit was predetermined to avoid age-related cognitive and visual decline. Exclusion criteria were: 1) any psychiatric or neurological comorbidity , 2) concomitant use of ADHD medications  4 weeks before entering the study, and 3) undergoing neurofeedback or cognitive training treatment.This was a two-month, open-label study in which eligible ADHD-diagnosed adults were provided with a pair of Neuro-glasses featuring a personalized visual stimuli pattern for each participant. A baseline assessment of the participants’ ADHD clinical profile pre-intervention included the Adult ADHD Self-Report Scale ASRS;  and the A follow-up assessment at the end of the intervention re-assessed ADHD performance on the ASRS and BRIEF-A questionaries. In addition, participants completed the CPT-3 test while wearing their Neuro-glasses.The Clinical Global Impression-Improvement CGI-I;  rating s™, supplied by VIZO Specs Ltd.) consisted of a standard eyeglasses frame and standard optic lenses with semi-transparent marking stimuli, a few millimeters in size, embedded on the lenses  sampling at 150 Hz with a 23.8-inch display mounted on top  and viewed at 60 cm distance. The tests were carried out using E-Prime 3.0  running on a standard personal computer . Eye-tracking parameters such as oculomotor reaction time and pupil size, as well as behavioral measures of time and accuracy, were collected and processed by VIZO’s proprietary cloud-based system to determine the optimal number and size of the stimuli and their positioning in the peripheral visual field.The outcome measures provide a broad assessment of ADHD symptoms, executive functions, global functioning, and measures of attention and impulsivity. The assessment involved self-report questionnaires, clinician evaluation, and a task-oriented computerized assessment.The Adult ADHD Self-Report Scale ASRS;  is a selThe Behavior Rating Inventory of Executive Function Adult Version BRIEF-A;  is a staThe Clinical Global Impression-Improvement CGI-I;  providesThe Conners’ Continuous Performance Test-3 CPT-3;  is a comTo assess adherence, participants were instructed to document their daily usage of the eyeglasses . Potential side effects and adverse events were assessed by the study investigators during the follow-up assessment.t-tests were used to test the effect of the Neuro-glasses intervention, comparing the baseline and end-of-intervention performances. Normality distribution of data was assessed using the Shapiro–Wilk test. Due to the exploratory nature of the study, no corrections for multiple testing were applied. The safety analysis included all available data on the intent-to-treat (ITT) population.Paired samples Mixed model ANOVA was used to explore any potential effect of covariates such as gender, age, eyeglasses, learning disabilities, treatment naïve, and ASRS total score. None of the covariates were found significant, thus were not included in the final model.D) using a paired t-test with a 0.05 two-sided significance level. Assuming a 20% attrition rate over the course of the study, 108 participants were targeted for inclusion  = 0.12, p = 0.037]. Self-reports on the ASRS total score approached significance , showing a 4% reduction in ADHD symptoms post-intervention. No significant effect was found on the ASRS hyperactive subscale . Improvement in the BRIEF-A was evident for the Metacognitive Index . There were no significant effects for the BRIEF-A Behavior Regulation index or the Global Executive Composite .t (92) = 2.25, p = 0.027) and reduced incorrect responses to non-targets  = 2.92, p = 0.004). All other CPT-3 measures were less sensitive to intervention-based changes in performance . Sixty-two percent of the participants met the response criteria, having much or very much symptomatic improvement after using the Neuro-glasses.No significant covariate effect was observed for demographic or baseline parameters such as gender, age, eyeglasses, learning disabilities, treatment naïve, and ASRS total score.This study provides preliminary support for the safety and efficacy of a peripheral visual stimulation-based device in managing ADHD in adults. Reduction in ADHD symptoms was demonstrated by the ASRS inattentive subscale, reflecting the rated adult’s difficulty in maintaining attention for long periods, organizing/planning, paying attention to details, and committing reckless mistakes . No signDespite the small effect sizes, the results are encouraging, given that the level of ADHD symptom manifestation in the study population was relatively mild, as reflected by the average ASRS baseline score. The clinical meaningfulness of the findings is further supported by the 62% of participants who positively responded to the intervention, as assessed by the CGI-I.Assessment of the intervention adherence rate revealed that most participants (63%) wore the Neuro-glasses for at least 4 hours daily, whereas only 7% reported wearing the Neuro-glasses occasionally for up to 2 hours daily. These findings, together with the relatively low dropout rate (<10%), are of high importance, as they indicate that the intervention was well tolerated and accepted by ADHD adults. Moreover, the high adherence rate suggests that the effect of the Neuro-glasses is notable and meaningful for the user.The current pilot study confirms that the Neuro-glasses hold minimal risks, as no clinically significant adverse events were associated with the intervention. Reports involved minor events of headache, dizziness, and eyestrain that were all transient, relatively mild and had not resulted in any discontinuations of the intervention.The etiology of ADHD is related to various genetic and environmental factors . Though The Neuro-glasses presumably attenuate ADHD symptoms by stimulating the visual periphery. However, it is still unclear what is the mechanism underlying their effect. A possible explanation is that the Neuro-glasses might operate via the arousal system. Arousal is crucial for survival, as it regulates consciousness, attention, alertness, motivation, and information processing. Arousal regulation reflects the competence of the brain to deal with ongoing situational demands. Its dysregulation has been linked to psychiatric and physiological disorders and cognitive impairments . RelevanWhile the Neuro-glasses are not an optical device by definition, they may operate via visual correction. A large body of evidence associates ADHD and problems of vision . ADHD paWhile the results of this study are encouraging, we acknowledge certain important limitations. First, this is an uncontrolled study. Thus, we cannot rule out the contribution of placebo effects. Second, given the exploratory nature of the study, no primary endpoint was predetermined. Third, the current study population does not represent the high prevalence of comorbidities in the ADHD population and their effect on attention symptoms since ADHD adults having any psychiatric or neurological comorbidity were excluded from the study. Fourth, this study does not control for the possible confounding effects of visual problems and emotional or motivational factors that may affect the conclusive assessment of improvement. Fifth, due to the study design, it is essential to recognize that with regards to the CPT specifically, we cannot rule out a possible acute impact of wearing the Neuro-glasses, as opposed to effects caused by their regular use.To conclude, Neuro-glasses provide a non-pharmacologically safe approach to managing ADHD in adults. These preliminary encouraging findings merit further research including future controlled studies evaluating the efficacy of a peripheral visual stimulation-based intervention in adults and possibly children with ADHD. Moreover, the current study provides important insights into the utility of visual stimulation as an intervention for treating ADHD and potentially for additional psychiatric and neurological disorders involving dysregulation of arousal levels.The original data collected in the study is included in the article. Further inquiries can be directed to the corresponding author.The studies involving humans were approved by Max Stern Yezreel Valley College Institutional Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.YR: Conceptualization, Methodology, Project administration, Resources, Supervision, Writing – original draft. CG: Conceptualization, Investigation, Writing – review & editing. GV: Conceptualization, Investigation, Writing – review & editing. CB-M: Data curation, Formal analysis, Methodology, Project administration, Writing – review & editing. DG: Conceptualization, Data curation, Methodology, Software, Writing – review & editing. NM-K: Supervision, Writing – original draft. ZE: Conceptualization, Investigation, Supervision, Writing – review & editing.


Previous diagnostic systems precluded the co-existence of autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD) in one person; but, after many clinical reports, the diagnostic criteria were updated to allow their co-occurrence. Despite such a clinical change, the neurobiological bases underpinning the comorbidity remain poorly understood, and whether the ASD+ADHD condition is a simple overlap of the two disorders is unknown. Here, to answer this question, we compared the brain dynamics of high-functioning ASD+ADHD children with age-/sex-/IQ-matched pure ASD, pure ADHD, and typically developing (TD) children. Regarding autistic traits, the socio-communicational symptom of the ASD+ADHD children was explained by the same overstable brain dynamics as seen in pure ASD. In contrast, their ADHD-like traits were grounded on a unique neural mechanism that was unseen in pure ADHD: the core symptoms of pure ADHD were associated with the overly flexible whole-brain dynamics that were triggered by the unstable activity of the dorsal-attention network and the left parietal cortex; by contrast, the ADHD-like cognitive instability of the ASD+ADHD condition was correlated with the atypically frequent neural transition along a specific brain state pathway, which was induced by the atypically unstable activity of the frontoparietal control network and the left prefrontal cortex. These observations need to be validated in future studies using more direct and comprehensive behavioral indices, but the current findings suggest that the ASD+ADHD comorbidity is not a mere overlap of the two disorders. Particularly, its ADHD-like traits could represent a unique condition that would need a specific diagnosis and bespoke treatments. Children with autism spectrum disorder (ASD) have cognitive rigidity and tend to persist in specific thoughts, whereas those with attention-deficit/hyperactivity disorder (ADHD) exhibit overly flexible cognition and have trouble with concentration. Despite such contrast, clinically, the two neurodevelopmental disorders often co-exist in one person. How can such a co-occurrence happen? By investigating the global and local brain dynamics, this study found that the comorbidity of ASD and ADHD is not a simple overlap of the two conditions. Instead, the cognitive instability seen in ASD+ADHD children was underpinned by unique brain dynamics that were not observed in pure ADHD. These findings indicate that the comorbid condition would need a bespoke diagnosis and treatment.Relations between autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD) have not been simple. ASD has cognitive rigidity as one of its core symptoms , whereasIn contrast to this major change in clinical practice, the behavioral property of the ASD+ADHD comorbidity is still under debate , and itsIn behavioral studies, the cognitive properties of ASD+ADHD individuals  have beeBiological knowledge of the brain mechanisms underpinning the ASD+ADHD condition is also limited. The neurobiological bases of pure ASD  and purePartly because of such behaviorally and biologically limited knowledge, some clinicians cast doubt on the concept of the ASD+ADHD comorbidity itself and warned that some standard medications for ADHD symptoms, such as the administration of amphetamine stimulants, might cause undesirable effects on ASD+ADHD individuals .Here, to address this situation, we aimed to identify the neural mechanisms underpinning the ASD+ADHD condition by directly comparing the global and local brain dynamics of high-functioning ASD+ADHD children with age-/sex-/IQ-matched pure ASD, pure ADHD and typically developing (TD) individuals .Technically, we first applied the energy landscape analysis  to restiThis case-control study analyzed rsfMRI data that were recorded from the following four types of cohort: children with ADHD only, those with ASD only, those with ASD and ADHD (ASD+ADHD), and TD controls. The datasets of the pure ADHD and corresponding TD children were shared on a website for the ADHD200 project , whereasWechsler Intelligence Scale for Children, Edition 4. The IQ scores of the pure ASD, ASD+ADHD and corresponding TD children were measured with the Wechsler Abbreviated Scale of Intelligence. The mean head motion was calculated in the preprocessing procedures of EPI data as described below.We selected participants based on their age (5 < age < 13), IQ , and head motion during echoplanar imaging (EPI) data recording (mean head motion ≤ 3 mm). We set the age range to reduce the effects of adolescence. The IQ scores of the pure ADHD and corresponding TD children were quantified by the ASD was diagnosed based on Autism Diagnostic Interview-Revised (ADI-R) and DSM-IV-TR. ADHD was diagnosed by clinical experts in accordance with Conners’ Parent Rating Scale-Revised (CPRS), long version.For the pure ASD group, we selected ASD children in the ABIDE dataset who met all the above conditions and had no comorbidity.For the pure ADHD group, we chose ADHD children with no other comorbidity in the ADHD200 dataset. We employed all the three types of ADHD  to ensure a sufficient sample size.For the ASD+ADHD group, we selected autistic children with ADHD from the participants enrolled in the ABIDE dataset. In the ADHD200 data, we found no ADHD children who showed ASD symptoms and met the above criteria for participant selection. In the ABIDE dataset, the comorbid ADHD in the ASD children was determined when the ASD children met all the requirements for ADHD except for criterion E in the DSM-IV-TR, which excluded the co-occurrence of ADHD in ASD individuals. In other words, we selected children who would be given ASD+ADHD dual diagnosis in the DSM-5.For the TD groups, we selected age-/sex-/IQ-matched TD children from the ADHD200 and ABIDE datasets, respectively.As a result, this study used the data recorded from 30 high-functioning pure ADHD children, 29 TD children for the pure ADHD group (TD in ADHD200), 33 high-functioning ASD+ADHD children, 30 high-functioning pure ASD children, and 38 TD children for the ASD+ADHD and pure ASD children , which were also provided in the ABIDE dataset, to evaluate autistic traits. The range of the ADI-R scoring system is sufficiently broad to evaluate the details of ASD symptoms; but the ADOS has narrower score ranges and, in particular, can give only a limited range of scores for cognitive rigidity . Given this, we used the ADI-R scores to assess the ASD symptoms.The severity of ADHD symptoms seen in the pure ADHD children was assessed based on the CPRS scores  that were provided in the ADHD200 dataset.The severity of ADHD-like traits of the ASD+ADHD group was not quantified with the CPRS scores since, as stated above, all the ASD+ADHD participants were selected from the ABIDE dataset, which did not contain the CPRS scores. Instead, here, we inferred such ADHD-like behaviors of the ASD+ADHD children based on the subscore for the repetitive restricted behaviors (RRB) in Autism Diagnostic Interview-Revised (ADI-R); that is, this study assumed that ASD+ADHD children with lower RRB scores should have more unstable/flexible cognition and manifest ADHD-like behaviors.t(61) = 2.1, p = 0.03 in a two-sample t test; This inference is primarily grounded on previous literature showing that (1) the symptoms of high-functioning ADHD individuals, in particular, their hyperactivity, are correlated with their cognitive flexibility/instability  and (2) In addition, we conducted a behavior experiment to provide supportive evidence for this rationale .All the MRI data were recorded in a 3T scanner  at New York University while the participants were asked to be relaxed with their eyes open. Functional MRI data were obtained using an echoplanar imaging (EPI) sequence . Anatomical images were recorded with a T1-weighted sequence .https://www.fil.ion.ucl.ac.uk/spm/software/spm12/) in the same manner as in our previous studies applying the energy landscape analysis or intrinsic neural timescale analysis to rsfMRI data (p > 0.4).We preprocessed the EPI data with SPM12 .The energy landscape analysis conducted here was essentially the same as that in our previous studies . Here, tTechnically, we first calculated the mean rsfMRI signal for each network at each time point in each participant. We then binarized the network activities using the whole-brain average fMRI signal as a threshold. This binarization procedure balanced the numbers of active and inactive states and should improve the accuracy of the following analysis . We repeUsing this dataset, we then conducted the energy landscape analysis. In short, first, we concatenated the binary data across participants in the same group and fitted a pairwise maximum entropy model (MEM) to them with a gradient ascent algorithm. We used the concatenated data so that we could achieve high accuracy of model fitting: in fact, because of the short fMRI scanning time , we could not fit the pairwise MEM to individual data with sufficient accuracy.This MEM fitting allows us to infer a hypothetical energy value for every activity pattern. Based on this energy value, we then built so-called dysconnectivity graphs C, which Theoretically, the group-level random-walk simulation is supposed to capture the brain state dynamics more accurately than the examination of the individual empirical rsfMRI data, because the numerical simulation can give a sufficiently long brain-state history for us to calculate all the types of brain-state transitions. In contrast, rare brain-state transitions are likely to be missed in the relatively short empirical rsfMRI data. Although, such an individual assessment of brain state dynamics based on each fMRI dataset is known to be somewhat correlated with the results of the random-walk simulation . Based ot was described such as i at time t .To quantify network-based brain state dynamics, we adopted a brain parcellation system  that divih is supposed to show the basal activity of network i, whereas i and network j. The i in the activity pattern kV and ih and ijJuntil the We then concatenated the binary data across participants in the same group and fitted a pairwise MEM to them. This simple model consisted of two parameters, D2) to that in the first-order model (D1) as follows (D1 – D2)/D1.The accuracy of this model fitting was evaluated by calculating a proportion of Kullback–Leibler (KL) divergence in this second-order model . In the graph, two activity patterns were regarded as adjacent if and only if their difference was seen at only one network activity.Next, we examined the structure of each group-specific energy landscape, which was defined as a graph of brain activity patterns N adjacent patterns. We then visualized and quantified the hierarchical structures between the local minima by building so-called disconnectivity graphs in the following procedures (N neighboring nodes. (2) We set a threshold energy level, Ethreshold, at the largest energy value among the 2N nodes. (3) We then removed the nodes whose energy values Ethreshold. (4) We examined whether each pair of local minima was still connected by a path in the slightly disconnected graph. (5) We repeated steps (3) and (4) after changing Ethreshold down to the next largest energy value. We stopped these procedures when all the remaining local minima were isolated. (6) Based on the obtained results, we built a hierarchical tree, disconnectivity graph, whose leaves  represented the local minima and internal nodes indicated the branching points of different local minima.In the energy landscape, we first searched for local energy minima, whose energy values were smaller than those of all the ocedures : (1) We i from the 2N nodes. If any of its neighbor nodes had a smaller energy value than the node i, we moved to such a node. Otherwise, we did not move and classified the node as a local minimum. We repeated this procedure until we reached any of the local minima. The initial node i was then assigned to the basin of the local minimum that we finally reached. This procedure was repeated for all the 2N nodes, which allowed us to classify all the brain activity patterns on the energy landscape, except for nodes on the saddles, into any of the basins. In this study, these basins were regarded as brain states  with a corresponding local minimum at their bottoms. First, we picked up node n states A.iV to move only to a neighboring pattern jV. Technically, we first chose one of such neighboring patterns randomly and then determined whether actual movement to the pattern occurred or not at the probability iV was more unstable than jV –B transition was significantly larger in the pure ADHD children than in the ASD+ADHD individuals  = 457.5, p < 10−5, Second, the indirect transition via either state C or D and either state E or F . In addition, this transition frequency was significantly higher in the ASD+ADHD children than in the pure ADHD individuals  = 158.8, p < 10−5, Third, we found that the indirect transition between states A and B via states C and F .Note that, for any of the three indirect transitions, no significantly different frequency was found between the two TD groups ; instead, the severity of their overall ADHD symptoms was correlated with their atypically enhanced A–[C/D–E/F]–B transition .In the pure ADHD group, by contrast, their symptoms were not explained by this A–C–F–B transition frequency  as a single region that showed a significantly shorter intrinsic neural timescale compared with the control group (t(57) = 4.9, pFDR < 0.05; F = 27.7, pBonferroni < 0.05, F = 15.5, pBonferroni < 0.05, In the pure ADHD children, we identified the left inferior parietal sulcus , dorsolateral prefrontal cortex  and inferior frontal gyrus  had significantly shorter neural timescales than the pure ASD group (t(61) > 4.1, pFDR < 0.05; t(69) > 4.8, pFDR < 0.05; In the ASD+ADHD children, the left superior frontal gyrus , to which the left IPS belongs. The mean neural timescale of the DAN was correlated with both that of the left IPS and the A–[C/D–E/F]–B transition frequency , which includes the left SFG. The neural timescale of the FPCN was associated with both that of the left SFG and the frequency of the A–C–F–B transition  and Oregon Health and Science University OHSU; .a–f) as in the original results.First, we confirmed that, in both datasets, the pairwise maximum entropy model was accurately fitted to all the types of participant data (≥87.5%), and the energy landscape analysis identified the same six brain states (States A–F) with the same six local minima  > 3.7, p < 0 0.05; p < 0.05; This overly frequent transition in the pure ADHD individuals was linked with the atypically short neural timescale of the left IPS  > 2.8, p < 0 0.05; p < 0.05; The A–C–F–B transition in the ASD+ADHD children was more frequent than in the other participant groups  close links between the ADHD symptoms and cognitive flexibility/instability  and (2) Psychologically, the current findings allow us to speculate cognitive differences between the hyperactivity of pure ADHD and cognitive instability of the ASD+ADHD comorbidity. Given that the DAN is closely linked to attention selection and generation of attentional sets , the cloClinically, this study implies the possibility of new noninvasive treatments for ASD, ADHD, and ASD+ADHD comorbidity. For example, as demonstrated in our recent study , some beIn addition, the current findings may indicate the necessity to re-evaluate the conventional treatments for high-functioning adults with both ASD and ADHD. In particular, given that the ADHD-like traits of ASD+ADHD individuals and those of pure ADHD patients are underpinned by different biological mechanisms F,G, we mThis neurobiological distinctiveness of the ASD+ADHD condition is also consistent with a recent systematic review of behavioral literature on the comorbidity : the revThe association between ASD symptoms and rigid brain state dynamics is consistent with and expands our previous findings . The priDespite intensive neuroimaging research on pure ADHD , the gloUsing the two data-driven analyses, the current study identified the distinct neural dynamics underpinning the ASD+ADHD comorbidity. The autistic social symptoms of the ASD+ADHD children were associated with similar neural bases as those of the pure ASD individuals, whereas their ADHD-like cognitive instability was governed by the unique global and local brain dynamics compared with the pure ADHD children. These observations indicate that “ASD+ADHD” is not a simple overlap of ASD and ADHD, and in particular, its cognitive instability would represent a distinct disorder and need unique treatments. The current approach focusing on the global and local neural dynamics could provide a new perspective for a comprehensive biological understanding of multiple neuropsychiatric disorders.