By using characterizations like XRD, FT–IR, FESEM, TEM, PL, and UV–Vis–DRS, the composition, structure, morphology, and optical absorption of samples were determined. The minimum inhibitory concentration (MIC) approach was used to investigate the antibacterial activity. Notably, this research indicated that nanofibers exert antibacterial action against both Gram-positive and Gram-negative bacteria with a MIC of 25 µg/mL. Furthermore, negatively charged E. coli was drawn to positively charged metal ions of Zr0.5Sn0.5TiO3/SnO2, which showed a robust inhibitory effect against E. coli. It was interesting to discover that, compared to pure TiO2, Zr0.5Sn0.5TiO3/SnO2 nanofibers revealed increased photocatalytic activity and exceptional cyclability to the photodegradation of Rhodamine B. The composite completely degrades dye in 30 min with 100% efficacy and excellent (97%) reusability. The synergetic effects of Zr0.5Sn0.5TiO3 and SnO2 may be responsible for increased photocatalytic and bactericidal activity.Environmental pollution is steadily rising and is having a negative influence on all living things, especially human beings. The advancement of nanoscience in recent decades has provided potential to address this issue. Functional metal oxide nanoparticles/nanofibers have been having a pull-on effect in the biological and environmental domains of nanobiotechnology. Current work, for the first time, is focusing on the electrospinning production of Zr E. coli and Enterococci are found in high numbers in the feces of animals; they are regarded as reliable indicators of potential damage to human health from fecal pathogens in fresh, untreated sewage [Environmental pollution is not a recent phenomenon, but it continues to be the greatest threat to mankind and a major factor of various illnesses and mortality. Worldwide urbanization, industrialization, mining, and excavation are human activities that have the greatest impact on the environment. Indisputably, water pollution is also a big concern in today’s world and a serious hazard that causes several problems for humans and the environment. Water pollution must be halted since it has been harming land and water species for years by entering lakes, rivers, streams, and seas. There are reports in the literature that an increasing number of individuals lack access to fresh, hygienic water. The water industry currently uses certain conventional chemical disinfectants, but it is alarming that many of these are carcinogens. Additionally, the need for more effective substitutes is growing as microorganisms’ resistance to conventional chemical disinfectants increases. d sewage . Furtherd sewage . Howeverd sewage . Pathoged sewage . The remd sewage . Water pd sewage . Nanotecd sewage . Recent d sewage . Nanopard sewage ,9. Recend sewage ,11. Furtd sewage . In compd sewage . Electrod sewage . Interesd sewage .2 is frequently regarded as the most ideal material for prevalent ecological uses because of its strong photoelectrochemical activity and stability against photo and chemical corrosion [2 surface using noble materials [The TiOorrosion . Howeveraterials ,17 and taterials .2–SnO2 system has been the focus of several investigations, including of semiconductors [2 works as a drain for photogenerated electrons when two semiconductor particles are linked [2 particle’s valence band, resulting in the proficient spatial parting of photogenerated charges and the suppression of recombination. Furthermore, the Sn dopant increases the photoinduced charge rate of TiO2 [2, SiO2, La2O3, etc., have also been utilized [2 to TiO2 improved the phase stability, surface area and ultimately photocatalytic effects [1−xSnxTiO4) thin films via the spin coating technique [2 demonstrates the potential mechanical properties of oxide ceramics. It is emerging as an important catalyst in a variety of photocatalytic and piezoelectric applications, and in ceramics production and dentistry for healing purposes [2 as an antimicrobial, anticancer, and antioxidant agent have been published. In this regard, Gupta et al. demonstrated the cytotoxic effect of ZrO2 nanoparticles on A549 cells and their antibacterial action on E. coli [2 structure to enhance its antibacterial action. Imran and colleagues published a study on the antibacterial properties of Fe3O4-doped ZrO2 nanoparticles. Their bactericidal effect against S. aureus, E. coli and B. subtilis has been increased by doping [2 has antibacterial action solely against E. coli, whereas ZrO2 complexes are active against both E. coli and S. aureus [2 nanoparticles against Staphylococcus strains. The findings showed their low activity at squat concentrations, and vice versa, both in the case of S. aureus and the mutant, respectively [Owing to the structural similarity between the two oxides, the TiOnductors . The cone linked ,20. Beca of TiO2 . To enhautilized ,15. Moreutilized ,23 are a effects ,25,26. Iechnique . Additiopurposes . Despite E. coli . There hy doping . Jangra . aureus . Anotherectively .2, ZrO2 and TiO2 in the current investigation, we aimed to design highly efficient Zr0.5Sn0.5TiO3/SnO2 (ZSTS) nanofibrous photocatalysts via the electrospinning method. Hitherto, there has been no published report regarding the synthesis, characterization and, more to the point, the bactericidal effect of these composite ceramics nanofibers. Herein, a first attempt was made to fabricate high-surface-to-area-ratio ZSTS ceramics nanofibers. Moreover, these high-aspect-ratio nanofibers have been investigated for their photocatalytic and antibacterial activities. These novel ceramics/metal oxide nanofibers will serve as the material of choice for environmental remediation, especially when aiming to control the quality of wastewater. Our research demonstrates that multifunctional ZSTS nanofibers may be a commendable candidate for use in health care and environmental pollution control systems.Conclusively, considering the noteworthy properties of SnOMw = 500,000 g/mol), tin(II)2-ethylhexanoate (98%), and zirconyl chloride octahydrate (98%). N, N-dimethylformamide  and titanium isopropoxide  were bought from Japan’s Showa Chemicals Ltd. And Junsei Company. E. coli and S. aureus were bought from ATCC to test the antibacterial activity. Unless otherwise stated, all the reagents employed were of analytical quality.Sigma-Aldrich from the United States supplied the polyvinyl acetate  was made by dissolving PVAc powder in DMF using magnetic stirring for 8 h at room temperature. In a separate vial, 5 g of titanium isopropoxide was mixed with a few drops of acetic acid until the mixture was transparent. The solution was then aggressively agitated while being gently blended with 6 g of PVAc solution. The resultant solution was put into a 10 mL syringe using a stainless steel needle. The counter electrode was a ground iron drum coated with a polyethylene sheet, and the positive terminal was a copper pin inserted into the solution from a high-voltage generator. In order to keep the solution in the capillary, the angle of inclination was adjusted. This solution received a 20 kV voltage. The distance between the collector and the needle tip of the syringe was set at 18 cm. The synthesized composite ceramics fiber mat was first dried at 80 °C for 24 h under vacuum before being calcined at 600 °C for 2 h in an environment of air at a heating rate of 2 °C/min.2.8H2O, 0.5 mM) in 3 mL of ethanol. The resultant mixture was electrospun and calcined under the same circumstances as those described for making titania nanofibers.Briefly, titanium isopropoxide  was continuously stirred into the PVAc solution with 1 mL of acetic acid. The solution was then supplemented with Tin(II)2-ethylhexanoate (1 m mole) and Zirconyl chloride octahydrate (ZrOCl−1. The photoluminescence (PL) spectra were obtained using a Hitachi, Japan, F-7000 fluorescence spectrophotometer with a 325 nm excitation wavelength.By using X-ray powder diffraction , the plain and ceramics/metal oxide nanofibers were identified for crystallinity. An energy-dispersive X-ray spectrometer (EDX) was used to test the chemical components of the products, while field-emission scanning electron microscopy  and transmission electron microscopy  were used to investigate the morphologies and structures. Thermogravimetric analysis (TGA) was performed in an air atmosphere from room temperature to 800 °C with a heating rate of 10 °C/min. On a UV–Vis spectrophotometer , the fibers’ UV–DRS (Ultraviolet diffuse reflectance) spectroscopy was recorded. A Nicolet Nexus 670 FTIR spectrophotometer was used to perform Fourier-transform infrared spectroscopy (FTIR) at a resolution of 4 cmThe rate of photodegradation of the ceramics nanofibers for Rhodamine B dye in deionized water was used to measure its photocatalytic activity. Following a typical procedure, 250 mL of Rhodamine B solution (20 ppm) was mixed with 0.1 g of photocatalyst using a magnetic stirrer for 30 min in the dark to reach an equilibrium between the dye and the catalyst before being exposed to UV light (λ = 254 nm). Every 5 min, a sample was taken, and to remove any potential photocatalytic particles, it was centrifuged. The degradation rate was then computed after the concentration of Rhodamine B was obtained via its UV–vis absorption at 553.5 nm .2 and ZSTS nanofibers. The aqueous solution containing TiO2 and ZSTS nanofibers was exposed to UV light for 30 min prior to being infused with bacteria, as described elsewhere [S. aureus and E. coli were grown in a Petri dish. To determine the minimal concentration for bacterial growth inhibition, the TiO2 and ZSTS nanofiber suspension was tested at several concentrations . Utilizing the earlier reported approach for the MIC, an initial evaluation of the antibacterial activity was conducted [6 CFU/mL, 5 mL of nutrient broth was injected with a loopful of overnight microbial cells from the two strains that were chosen from the agar plates. As previously defined, pure TiO2 and various concentrations of the ZSTS nanofibers were irradiated using UV and tested. An Ultraviolet (UV)–Visible spectrophotometer  was deployed to measure the optical density (OD), which was recorded every three hours during an incubation period.The antibacterial activity was scrutinized using a conventional process by exposing pathogenic organisms cultivated at pH 7.4 and an rpm of 150 to different concentrations of TiOlsewhere . Bacterionducted . BrieflyThe one-dimensional nanometer-scale shape and the distinctive chemical and electrical characteristics of metal oxide nanofibers have attracted a great deal of scientific attention. It has been shown that they have a range of uses in light-emitting diodes, gas sensors, liquid crystal spectacles and cosmological cells. A quick and simple approach to creating nanofibers with a diameter many orders of scale smaller to those made via traditional spinning procedures is electrospinning. Electrospun nanofibers have several uses in photocatalysis, filtration, protective fabrics, battery cells, and biomedicine . The pot2 nanofibers and the ZSTS nanofibrous composite has been investigated via XRD, as demonstrated in 2 (JCPDS No. 89−4921). Meanwhile, all the diffraction peaks shown in spectrum b of 0.5Sn0.5TiO3 (JCPDS No. 34−0033), and the tetragonal structure of SnO2 (JCPDS No. 71-0652). Each peak of SnO2 corresponds to the (110), (101), (200), (211) and (301) plane [0.5Sn0.5TiO3 and SnO2 indicate their commendable crystallinity. No traces of other phases are examined, confirming the high purity of the samples. Thus, it is confirmed that the material formed is a fibrous ZSTS nanocomposite.The crystal structure report associated with TiO1) plane . The sha2 and ZSTS composite nanofibers through different magnifications. 2 nanofibers is quite smooth and has a diameter of around 200 nm, which is also confirmed by the TEM image  could be defined as a metal–oxygen (M–O) stretching vibration [In the IR spectra of TiO visible a. Characi–O bond . Figure ibration .2 photocatalyst. The presence of zirconium and tin oxide, which have a wider band gap than titania, may be responsible for the shift in the intensity of the light absorption in the composite sample. To estimate the bandgap energy (Eg) of the samples, Eg was calculated from the plot of (αhν)1/2 versus the energy of the stimulating light (hν) using equation αhν = A(hν − Eg)n/2 [2 and ZSTS nanofibers were determined to be 3.28 and 3.45 eV, respectively (inset The absorption of light obtained from the diffuse reflectance UV is displayed in − Eg)n/2 . As a rely inset .2. The strength of the PL peaks, which are connected to the recombination of the electron–hole pairs within the semiconductor, further suggests that there is an effective charge transfer within the ZSTS nanofibers. The photogenerated electron–hole pairs can readily transfer at the heterostructured interface in the case of the ZSTS composite, leading to greater photocatalytic activity when exposed to UV light.2 and ZSTS composite was assessed using the photodegradation of RhB dye under UV light irradiation, as shown in 2 decolorizes RhB by 70% in 30 min. The ZSTS composite nanofibers, however, show a noticeably improved photocatalytic performance. The composite sample demonstrates that all of the dye can be broken down within 30 min. The experimental findings showed that ZSTS composite photocatalysts have greater photocatalytic activity than pure TiO2 photocatalysts. This improvement in photocatalytic efficiency could be due to the heterostructure that forms when Zr0.5Sn0.5TiO3 and SnO2 come together, since it is crucial for the separation of electron–hole pairs. Therefore, it seems more likely that the superior photocatalytic activity of ZSTS is attributable to a more effective separation of the electron–hole pairs or to a significantly reduced propensity for electron and hole recombination.The photocatalytic activity of the pure TiO2 with SnO2 increases their photocatalytic activity. Owing to the charge separation that takes place at the interface between two semiconductors, this improvement is attributed to a drop in the rate of electron–hole recombination [4+ ions in the TiO2 lattice increases the parent oxide’s exterior acidity [2/ZrO2 photocatalyst, which results in the detection of more O2H• radicals. However, in the case of TiZr, the heteroatom takes part in the electron trapping sites that result in the establishment of Zr4+–O2•– species, and this possibly has an impact on the inherent photoactivity of matter [Numerous studies have also documented how combining TiObination . In addi acidity , which mf matter .To evaluate the reusability of the composites from the standpoint of practical applications, the ZSTS nanofibers were selected. The catalysts currently in use are easily separated from the solution and effectively dispersed when stirred. Centrifugation makes it easy to separate the catalyst from the cleansed solution. The dye did not adhere to the catalyst permanently; rather, photodegradation is what causes the cleaning. The ZSTS composite showed good catalytic stability, keeping a similar level of reactivity even after five cycles (97%) . The una2 and ZSTS composite nanofibers against two typical bacteria was demonstrated and is shown in E. coli, which frequently causes severe diarrhea, and S. aureus, an opportunistic pathogen that frequently causes respiratory infections and food poisoning, are the bacteria that were studied. By treating them using ZSTS nanofibers and the pure TiO2, bacterial growth as an indicator of turbidity was observed and computed in order to assess their antibacterial capabilities. The outcomes attained point out that the ability of TiO2 nanofibers to inhibit the growth of bacteria is exclusively associated with its selected concentration and UV irradiation, comparable to previous outcomes [2 organization, investigators have proposed diverse elimination mechanisms, including negative effects on deoxyribonucleic acid, membrane impairment that causes the cell contents to leak out, and observed reductions in respiratory operations owing to the loss of precursor enzymes [2 solution was exposed to UV radiation. A hole (h+) is produced in the valence band via charge separation, while an electron (e−) is produced in the conduction band. Hydroxyl radicals (OH) are produced at the surface of the excited particle by elusive electrons in the valence band holes of water and/or hydroxyl ions. The superoxide anion (O−2) is created when electrons reduce oxygen. The ROS produced by UV-irradiated TiO2 nanofibers may have initially interacted with the cell wall, produced free radicals that can enter into the interior of the cell, disrupt the internal contents and cause the cells to deform, leading to disorganization and leakage [2 nanofibers, the ZSTS nanocomposites demonstrated action against bacterial growth that was more severe (E. coli. Additionally, it was discovered that ZSTS nanocomposites and TiO2 nanofibers make E. coli more sensitive. The two strains’ different cell structures have been shown to be the cause of increased sensitivity [S. aureus, a Gram-positive bacterium, has a dense covering of peptidoglycan that prevents nanoparticles from easily entering S. aureus. It could be interpreted that in the case of Gram-negative bacteria, the desired outcome may be achieved via the electrostatic attraction between negatively charged bacteria and positively charged metal ions, which causes bacteria to rupture and die. A similar interpretation of ZrO2 nanoparticles has been reported elsewhere [Antibiotic resistance has incurred staggering health care expenditures in the community over the last decade, necessitating immediate action. This demands a thorough examination of the innovative possibilities. With the ability to autonomously control and manipulate both atoms and molecules, nanotechnology is a multidisciplinary field of study that has enabled significant advancements in numerous scientific fields ,44. In toutcomes . In UV/T enzymes . Neverth leakage . In the e severe b. The cusitivity ,46. The lsewhere .2 and ZrO2 nanoparticles against infections [2 NPs in tooth decay assertions were identified [Previous researchers have documented the bactericidal effects of cotton treated with ZrOfections . In yet entified . Accordientified . As such2 and ZSTS nanofiber photocatalysts were prepared via the electrospinning method. The experimentation evaluating the photodegradation of RhB and PL investigation revealed that ZSTS nanofibers have a noteworthy effect on the separation of photogenerated charge carriers and UV light photocatalytic action. Furthermore, in 30 min, the composite completely bleached the colorant. The produced photocatalysts also exhibited stability when exposed to UV light. The synergetic effects between Zr0.5Sn0.5TiO3 and SnO2 were mostly responsible for the increased photocatalytic activity of the ZSTS composites. The above findings contribute a very effective and robust photocatalytic material for eliminating biological and chemical pollutants, in addition to offering updated knowledge on how to develop some effective ceramics/metal oxide nanofibrous photocatalysts. Interestingly, the ZSTS demonstrated efficient antimicrobial potential for both Gram-negative and Gram-positive bacteria; however, due to their negatively charged cell surface and scavenging activity, these photocatalysts were more vigorous against Gram-negative bacteria. Despite the substantial advancements, there are still a number of problems associated with antibacterial nanofibers that need to be resolved. First, it is necessary to attempt the manufacturing of composite antibacterial nanofibers on a large scale, as it is currently primarily restricted to the laboratory, and to investigate the biocompatibility of these high-aspect-ratio nanofibers with human cell lines. Therefore, the ZSTS could act as new material for environmental applications.In summary, TiO


This study calls attention on molecular mimicry and the consequent autoimmune cross reactivity as the molecular mechanism that can cause adverse events following meningococcal B vaccination and warns against active immunizations based on entire antigen. Neisseria meningitides(aka meningococcus) can cause a multitude of severe illnesses, collectively termed meningococcal disease.The bacteriumImmune system disorders.Hypersensitivity reactions, including anaphylactic reactions.Nervous system disorders.Syncope.Currently, at the best of the author's knowledge, no investigation/hypothesis has been proposed to define the molecular mechanism that triggers the adverse events following the Trumenba vaccine administration. In analyzing the issue, this study posed the question of whether molecular mimicry between the vaccine antigen and the human proteins might play a role as already found in analogous research models.https://www.uniprot.org/uniprotkb/Q9JXV4/, was dissected into pentapeptides offset each other by one aa residue  and the resulting bacterial pentapeptides were analyzed for occurrences within the human proteome.Molecular mimicry between the bacterial fHbp antigen and human proteins was analyzed according to published methodology.www.uniprot.orgPentapeptides were used since a peptide grouping composed of 5 aa defines a minimal immune unit that can (1) induce highly specific antibodies and (2) determine antigen–antibody specific interaction.Following molecular mimicry analyses, it was found that fHbp pentapeptides repeatedly occur for a total of 2,809 multiple occurrences in human protein alterations which can lead to severe diseases. The bacterial versus human pentapeptide overlap is of such a dimension that obviously it cannot be reported in the context of an article. Consequently, data are reported asIn parallel, the severe diseasesThe vast sharing of immune peptide determinants between the bacterial fHbp antigen and the human proteins warns against Trumenba utilization to prevent meningococcal diseases. Indeed, the present data speak for themselves and clearly predict a very high incidence of autoimmune pathologies in the vaccinees as a result of molecular mimicry and the consequent potential cross-reactivity, thus factually confirming what had already been stated in the prescribing information of the Trumenba vaccine.In synthesis, this report adds to numerous previous studies,


Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, and Mycobacterium tuberculosis [Antimicrobial-resistant bacterial infections pose a significant challenge to health worldwide. In 2019, there were an estimated 1.95 million deaths and 47.9 million lost disability-adjusted life-years attributable to antimicrobial resistance (AMR) [rculosis . TargeteAntibiotics are not being developed at the speed necessary to keep up with the evolution of bacterial AMR, with only 13 new drugs in Phase III clinical trials as of a March 2021 report . More thCorynebacterium diphtheriae, Neisseria meningitidis, Haemophilus influenzae type b (Hib), and Bordetella pertussis resulted in notable decreases in cases [S. pneumoniae (including antibiotic-resistant isolates), demonstrating vaccination combating AMR in action [Vaccination is an integral strategy to prevent AMR, and reduces infections caused by both antibiotic-susceptible and -resistant bacteria, thereby reducing the overall usage of antibiotics. The success of vaccines in reducing disease burden over the last century is clear; vaccines against in cases . The intn action . Beyond n action . Vaccinen action . This alK. pneumoniae was responsible for the largest portion of deaths attributable to AMR, at 19.9% of deaths attributed to AMR [As the global burden associated with AMR grows, interventions must be directed towards populations at highest risk of infection and death, including those in low- and middle-income countries (LMICs), immunocompromised and other individuals with comorbidities, and children, who are often excluded from studies investigating novel antimicrobials. Preventing infections is key; in regions, limited access to antibiotics and/or lack of strong antibiotic regulation challenges successful treatment of drug-sensitive infections . In 2019d to AMR .E. coli infection with a larger bacterial burden [E. coli burden in the bladder [E. coli contribute to the most deaths of any resistant bacterium worldwide and the increased risk of E. coli infection in people with diabetes is just one example of a scenario where vaccines against resistant bacteria should be prioritized for specific groups.Individuals with comorbidities have higher risk of infection not only as a direct result of their disease and treatments of it, but through increased hospital exposure, frequent invasive procedures, and as a result, increased antimicrobial usage . Diabetel burden . People  bladder . Drug-reSpecial formulations of antibiotic drugs intended for pediatric use are often developed after the antibiotic has been marketed in adults, leaving children behind in accessing effective antibiotics. Pediatric antimicrobial development includes several hurdles, with different dosages and/or formulations needed that lead to technological and regulatory challenges . Yet, itK. pneumoniae, a major cause of sepsis with increasing rates of AMR [S. pneumoniae) may not be feasible [M. tuberculosis (TB) for nearly a century and is effective in protecting very young children [While vaccination is an optimal strategy to combat AMR in many ways, bacteria can challenge vaccine development through their high genetic diversity, a range of potential antigens and the ability to cause a variety of infections with differing outcomes depending on the status of the host. The genetic heterogeneity of many bacterial pathogens leads to a diversity of structures in protein and polysaccharide antigens, rendering vaccine development difficult and strain cross-protection unlikely . Multicos of AMR . With mofeasible . Bacilluchildren . HoweverSeveral approaches exist in bacterial vaccines that are currently included in routine immunization schedules, including whole inactivated bacteria, live-attenuated, and subunit vaccines including toxoid and conjugate vaccines. These approaches can include monovalent or multivalent formulations targeting more than 1 antigen. Other platforms have been investigated in recent clinical studies, including the application of nanoparticles, such as outer bacterial membrane vesicles (OMVs), and nucleic acid vaccines that may include DNA-based plasmids or RNA-encoding target antigens . The intS. pneumoniae and H. influenzae, respectively [E. coli (ExPEC) [N. meningitidis and is being used for other vaccines and could help address the high diversity of AMR strains.Current bacterial vaccines are demonstrably effective at reducing disease burden and consequently address AMR via reduction of antibiotic usage. Both pneumococcal and Hib vaccines target the capsular polysaccharides (CPS) of their bacterial species, ectively . The pneectively . Thus, t (ExPEC) . BioinfoSalmonella enterica Typhimurium, Hib, and S. pneumoniae. Vaccines that are currently being tested in Phase III clinical trials include those against extraintestinal pathogenic E. coli, Salmonella enterica ParaTyphi A, and N. gonorrhoeae. These vaccines target bacteria that have increasing rates of resistance and will reduce both susceptible and resistant infections. Unfortunately, based on the current lack of progress in vaccine development, the WHO has identified several resistant pathogens for which vaccine development is currently thought to have lower feasibility and recommends resources be focused on other tools for the prevention and control of AMR in these bacteria.As we look to the future, there are several vaccines in the global vaccine development pipeline that the WHO has identified as having high potential to reduce global AMR . This inAMR is one of the most complex and pressing issues facing modern medicine; vaccines will be a critical part of our strategy to combat rising AMR-associated morbidity and mortality and will be more successful against some pathogens over others. Most vaccines that protect against bacterial infections target common antigens such as the capsular polysaccharide. Moving forward, newly developing technology in vaccine platforms and novel antigen discovery should continue to be explored in preclinical and clinical models. The scientific community must maintain equitable production and distribution as a fundamental element of the vaccine development pipeline. This will ensure that vaccines targeting bacterial AMR will reach the populations who need it most.


Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was investigated, and the samples loaded with ɛ-PL demonstrated stronger antimicrobial activity against S. aureus.In the present study, electrospun nanofiber mats were fabricated by mixing different ratios  of Persian gum (PG) and poly (ethylene oxide) (PEO). The SEM micrographs revealed that the nanofibers obtained from 93% PG and 7% PEO were bead-free and uniform. Therefore, it was selected as the optimized ratio of PG:PEO for the development of antimicrobial nanofibers loaded with ɛ-Polylysine (ɛ-PL). All of the spinning solutions showed pseudoplastic behavior and the viscosity decreased by increasing the shear rate. Additionally, the apparent viscosity, G′, and G″ of the spinning solutions increased as a function of PEO concentration, and the incorporation of ɛ-PL did not affect these parameters. The electrical conductivity of the solutions decreased when increasing the PEO ratio and with the incorporation of ɛ-PL. The X-ray diffraction (XRD) and Fourier-transform infrared (FTIR) spectra showed the compatibility of polymers. The antimicrobial activity of nanofibers against  Streptococcus albulus, which is decomposed to lysine, and can be considered a source of lysine amino acid [Amygdalus scoparia Spach) shrubs which are grown in the central regions of Iran [The microbial contamination of food products may cause spoilage and foodborne diseases. These pathogens have posed concerns in the food industry and to public health. Changing the sensory properties of food products and increasing the risk of hospitalization and death are the major adverse effects of food pathogens ,2. Foodbino acid . ɛ-PL haino acid . Other iino acid . On the ino acid . Electroino acid . Moreoveino acid . Therefo of Iran . It is a of Iran ,15. Howe of Iran . A combi of Iran  revealed of Iran . This po of Iran , cellulo of Iran , chitosa of Iran , and whe of Iran  to make S. aureus and E. coli, two of the most important and widely spread food pathogens, were investigated.However, to the best of our knowledge there is not a published paper about the development of PG/PEO fiber mats. Therefore, in this study, antimicrobial electrospun fiber mats were fabricated using different ratios of PG and PEO . Subsequently, ε-PL was added to the optimal ratio and the physicochemical and antimicrobial properties of fiber mats against 6 Da. Poly (ethylene oxide) (average molecular weight = 9 × 105 Da) and ε-Polylysine were acquired from Sigma-Aldrich . The nutrient agar culture was obtained from Merck Chemicals Co. . E. coli and S. aureus were procured from the Persian Type Culture Collection (PTCC).Persian gum was collected from wild almond shrubs in Shiraz, Iran. It contained 7.92% moisture, 2.21% ash, 0.18% fat, 0.22% protein, 87.88% total sugar, and 9.68% uronic acid. The average molecular weight of Persian gum was 4.15 × 10w/v) was prepared and placed on a magnetic stirrer at room temperature for 24 h to be completely hydrated. PEO was added to distilled water to prepare a 5% w/v solution and magnetically stirred at 70 °C for 30 min. Then, different ratios of PG:PEO  were mixed for 30 min with a magnetic stirrer at room temperature to obtain the spinning solutions. Finally, ε-PL(1 mg/mL) was added to the electrospinning solution of the sample, which produced the optimal nanofibers, and it was stirred for 5 min.A solution of PG in distilled water (5% −1. The dynamic oscillatory measurements were performed with a cone and plate geometry (1 mm gap and 25 mm cone diameter). The spinning solutions were poured into the rheometer cup and covered with silicon oil to prevent the evaporation of water during the experiments. Afterward, frequency sweeps tests were carried out within the frequency range of 0.1 to 100 Hz at 0.5% strain and 25 °C.The rheological properties of spinning solutions were measured with a rheometer . Steady shear data of spinning solutions were obtained at 25 °C, and shear rates were from 14 to 300 SThe electrical conductivity (EC) of the spinning solutions was determined with a conductivity meter  at 25 °C. Two electrodes with the same voltage were placed in the solution, and the current that it created, based on the conductive nature of the solution, showed the conductivity of the device.Nanofibers were fabricated with an electrospinning apparatus  at 22 °C and 40% relative humidity. The instrument consisted of a high-voltage supply, a syringe pump, and a rotating collector. A 3 mL syringe with an 18-gauge needle was filled with the electrospinning solution and placed in front of the flow pump. The drum collector was covered with aluminum foil to collect the fiber mats. The spinning parameters were as follows: spinning voltage of 15 kV, injection rate of 1.3 mL/h, and receiving distance of 10 cm.To investigate the morphology of nanofibers with a scanning electron microscope, the resulting fibers were coated with gold using a sputter coater  and photographed with an SEM device  at a voltage of 20 kV and a magnification of 3000.−1.To identify the functional groups, chemical bonds, and molecular interactions, the FTIR spectra of nanofibers were obtained with an FTIR spectrometer  with a wavelength resolution of 4 cm within the range of 4000 to 400 cmThe crystal structure of nanofibers was investigated using an X-ray diffractogram  with a diffraction angle of 5 to 60 degrees, a voltage of 40 kV, and a current of 40 mA.Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The bacterial inoculums (0.5 McFarland standard or 1.5 × 108 CFU/mL) were cultured on nutrient agar plates. Round-shaped fiber mats (7 mm in diameter) were cut under sterile conditions and placed on inoculated plates. The plates were incubated for 24 h at 37 °C, and then the inhibition zone diameter around the nanofiber discs was determined with a caliper.The antibacterial properties of the PG/PEO nanofibers with and without ɛ-PL were investigated against two food pathogenic bacteria, p < 0.05 was considered significant.The experiments were replicated three times, and the obtained data were reported as mean ± standard deviation. All statistical analyses were accomplished by the SPSS software . The data were submitted to an analysis of variance (ANOVA) and Duncan’s multiple range test, and The primary experiments revealed that pure PG solution is not spinnable. This behavior could be due to the strong repelling forces between biopolymer molecules that prevent the formation of sufficient chain entanglement. To increase the spinnability of PG, different ratios of PEO were blended with PG. PEO is a water-soluble, nontoxic, biodegradable, and biocompatible synthetic polymer that decreases the repulsive forces between molecules, modifies the electrical conductivity and viscosity of the spinning solution, and promotes the electrospinning process . The lowThe solution comprising 93% PG and 7% PEO was selected as the optimal electrospinning solution to produce the ɛ-PL-loaded PG/PEO nanofibers. The scanning electron micrographs of PG/PEO nanofibers with ɛ-PL revealed that the addition of ɛ-PL did not affect the spinnability of the fibers.The viscosity of the polymeric solution is an important factor in electrospinning because it has substantial effects on the formation of chain entanglement of the polymer molecules, which is required for the successful electrospinning of biopolymers . As showThe elastic (G′) and viscous (G″) moduli of the spinning solutions are presented in The electrical conductivity of electrospinning solutions is a key factor in the fabrication of lead-free and uniform fibers. Sufficient electrical conductivity can overcome the surface tension of electrospun droplets, increase the electrostatic repulsion, facilitate the jetting process, and produce uniform fibers in electrospinning , while h−1 corresponds to the hydroxyl groups. The peak at 2927 cm−1 is related to the asymmetric stretching of CH3, while the band at 2859 cm−1 corresponds to the symmetric CH2 groups. The strong band at 1608 cm−1 is the indicator of COOH groups in uronic acid residues [−1 is related to the bending vibration of C–H. The peaks between 1200 and 1000 cm−1 are assigned to the C–O and C–C complex bands. The absorbance band at 1017 cm−1 is attributed to C–O or C–C functional groups, and the peak between 1000 and 700 denotes C–H vibrational bonds. The characteristic peaks of PG were observed in the FTIR spectra of nanofibers, and the peak at 1017 was intensified by the increase in the PEO ratio.FTIR analysis was performed to study the compatibility of spinning polymers with each other. The FTIR spectra of nanofibers are presented in The crystallographic structures of PG/PEO nanofibers with and without ɛ-PL are presented in S. aureus and Gram-negative E. coli. E. coli. and S. aureus. This indicates the ability of ɛ-PL to inhibit bacterial growth. These findings are in line with the results reported by Lin et al. [E. coli. was 13 ± 0.5 mm, while it was 17 ± 0.5 mm for S. aureus, indicating that the Gram-negative E. coli. was more resistant to ɛ-PL than the Gram-positive S. aureus. The Gram-negative bacteria have a lipopolysaccharide outer membrane, which may have a protective effect against antimicrobial agents. These findings support the findings of Li et al. [The antimicrobial activity of ɛ-PL-loaded fiber mats versus control nanofibers was investigated against two common foodborne pathogens, Gram-positive n et al. , who repn et al. . AdditioStaphylococcus aureus and Escherichia coli, two important food pathogens. Therefore, PG/PEO/ɛ-PL nanofiber mats have good potential for active food packaging.In conclusion, the nanofiber mats were successfully produced from PG and PEO using the electrospinning technique. The experiments revealed that PEO is compatible with PG for electrospinning and can be considered a suitable polymer for making PG solution blends for electrospinning. A low concentration (7%) of PEO is enough to produce bead-free and uniform nanofibers. It was also found that antimicrobial fiber mats can be produced by the incorporation of ɛ-PL. These nanofibers could prevent the growth of


Patterning is attractive for nanofabrication, electron devices, and bioengineering. However, achieving the molecular-scale patterns to meet the demands of these fields is challenging. Here, we propose a bubble-template molecular printing concept by introducing the ultrathin liquid film of bubble walls to confine the self-assembly of molecules and achieve ultrahigh-precision assembly up to 12 nanometers corresponding to the critical point toward the Newton black film limit. The disjoining pressure describing the intermolecular interaction could predict the highest precision effectively. The symmetric molecules exhibit better reconfiguration capacity and smaller preaggregates than the asymmetric ones, which are helpful in stabilizing the drainage of foam films and construct high-precision patterns. Our results confirm the robustness of the bubble template to prepare molecular-scale patterns, verify the criticality of molecular symmetry to obtain the ultimate precision, and predict the application potential of high-precision organic patterns in hierarchical self-assembly and high-sensitivity sensors. An efficient bubble-template printing method is developed for fabrication of nano- and molecular-scale patterns. Patterning holds great promise for application in nanofabrication  molecular-scale ultrathin films with a controllable number of layers at the gas-liquid interface  approach by introducing the ultrathin liquid film of bubble walls as a soft confinement space. Using a two-fragment molecular modal system, patterns with width up to sub–12 nm are achieved, which consisted of six layers of molecules. We point out that disjoining pressure isotherm curve could be used to predict the highest pattern precision for various model molecules. The aggregation-induced emission (AIE) π-conjugated core structure capably enables the in situ visualization of the bubble evolution process, especially the bubble rupture stage, which is notable for understanding the diversity and uniformity of pattern morphologies. Furthermore, we experimentally and theoretically reveal the molecular assembly mechanism in the bubble wall confinement space and demonstrate that the symmetric molecules exhibit much better reconfiguration capacity and smaller preaggregates, which are key points to stabilize the foam film drainage and construct higher-precision bulk phase–dominant patterns. It could be expected that asymmetric structures are more favorable to reaching the ultimate bilayer molecular precision due to the denser film at the gas-liquid interface. These findings provide a powerful solution for 1D molecular assembly, as well as ultrahigh-precision patterning and multiple-functionalized application.TPE-diSDS (fig. S2) is chosen, which retains the surface activity characteristic from the sodium sulfonate group  to ensure the stability of bubbles while inheriting the AIE on-state property from the tetraphenylethene group  to facilitate the in situ visualization. Last, the operation of molecular printing is simply conducted with a sandwich structure on the arbitrary targeting substrate , which makes bubble template a universal and scalable strategy for molecular patterns  can accurately distinguish this stage from the latter two because the molecular concentration remains unchanged during the gas transport process at a time scale of 6 to 8 min . The phase diagram of TPE-SDS (fig. S10) indicates that it follows similar regulation rules for precision control to that of TPE-diSDS. However, the highest precision can only reach 50 nm, much lower than that of TPE-diSDS.On the basis of the two-fragment molecular strategy, the BTMP method realizes ultrahigh-precision patterns at the molecular level. Through statistical analysis of the molecular pattern widths under different printing conditions, controllable precision can be achieved from ten to several hundred nanometers  shows thTPE-SDS, the foam film drains rapidly and finally ruptures around 50 nm  in foam physics , indicating that it is no longer characteristic of a well-layered stacking. The results reveal that the BTMP method can induce molecular ordering at the micro/nanoscale with desirable structural characteristics. Furthermore, the molecular dynamics (MD) simulation of the BTMP process shows the difference in the assembly behavior of different molecules near the dried state (movie S2). Moreover, TPE-diSDS with symmetric structure exhibits good reconfiguration capability with the contraction of bubble wall confinement space and keep a stable lamella stacking mode during the assembly process, ranging from wet state, drying state, to dried state , it is expected to achieve the ultimate Newton black film thickness in the future.We have developed an efficient bubble-template printing method for the fabrication of nano- and molecular-scale patterns. Using the template of the bubble arrays, we can precisely confine the molecular assembly and achieve various morphologies with sub–12 nm precision. In particular, we demonstrate that the highest precision of BTMP could be predicted by the disjoining pressure isothermal curve, and the exact value is closely correlated with the molecular structure. In addition, we clarify the confinement-induced molecular aggregation and assembly mechanism for molecules with different symmetries. It is revealed that symmetric molecules with better reconfiguration capacity, smaller aggregates, and anisotropic lamellar structure are more favorable to construct the few-layer level molecular patterns. However, because the ordering of the interface phase will play a more apparent influence rather than that of the bulk phase when it comes to the Newton black film precision, the asymmetric molecules hold more hope for approaching the ultimate bilayer precision. These findings afford powerful solutions for ultrahigh-precision molecular patterning and provide promising opportunities for the fabrication of nano- and molecular-scale devices, especially high-sensitivity sensors (fig. S17), photoelectric devices with unique nanoconfinement and performance enhancements, and bioengineered tissues.TPE-diSDS and TPE-SDS were synthesized referring to earlier reports  limits the shrinkage of liquid into the gap between the template and the substrate, thus forming these line morphologies affected by the template. Besides, the broken line 2 also requires low concentration to realize discontinuous morphology.The processing of the template with columnar microstructure has been described in the previous article (The structure of the molecular patterns was investigated by SEM (SU-8020 and S-4800) and TEM (JEM-1011). Optical microscopy images and cross-polarized optical microscopy were obtained by Nikon . Static CAs were measured on a DataPhysics OCA20 CA system at the substrate. The experimental results of fluorescence visualization and temperature sensor were completed using confocal microscope C2 and A1 produced by Nikon Company. The 3D image results were achieved by layer-by-layer scanning and 3D reconstruction. In addition, the wavelength of exciting light was 405 nm, and the detection wavelength of 450 to 600 nm was selected. White light interferometric imaging of printed molecular patterns was characterized by surface profile instrumentation from Bruker Company. AFM results were also achieved with Bruker’s Multimode 8 AFM. Cu Kα source x-rays was used in XRD . The spatial polarization profile of light emission for the solid patterns was characterized by the excitation of mercury lamp and the detection of luminescence information from different angles by the linear polarizer. Nuclear magnetic resonance spectra were recorded by Bruker Avance (400 MHz) spectrometer. Mass spectrum was measured on matrix-assisted laser desorption/ionization–time-of-flight liquid chromatography–mass spectrometry of Bruker. Fluorescence spectra were taken on a Horiba spectrofluorometer (FluoroMax), and the excitation wavelength was set to 310 nm. Ultraviolet-visible (UV-vis) spectrum was taken on a UV-vis spectrophotometer . Surface tension of the solutions was measured through Wilhelmy hanging plate method and BZY-2 tensiometer.22H43O2) standard. The 1D intensity profiles were integrated from background-corrected 2D WAXS patterns.GIWAXS were measured on a Xeuss 2.0 SAXS/WAXS system . Cu Kα x-ray source (GeniX3D Cu ULD), generated at 50 kV and 0.6 mA, was used to produce x-ray radiation with a wavelength of 1.5418 Å. A semiconductor detector  with a resolution of 487 × 619 pixels (pixel size = 172 μm by 172 μm) was used to collect the scattering signals. The incident angle was 0.3°. Each WAXS pattern was collected with an exposure time of 30 min. The sample-to-detector distance was 137.5 mm, which was determined by a silver behenate , green (546 nm), and blue (470 nm); I is the intensity measured in each pixel; Imax and Imin are the maximum and minimum intensity values; n is the refractive index; and R is the Fresnel coefficient of the solution. Increasing the pressure inside the cell to change the thickness of film can thereby get the entire disjoining pressure isotherm curve.The disjoining pressure isothermal curve was measured by a homemade Scheludko-like cell (Cryo-TEM images were acquired by a Themis 300 (Thermo Fisher Scientific). For the sample preparation, a total of 3 μl of solution were deposited onto a holey carbon film on copper grid [grid in grid (GIG)] and flash-frozen in liquid ethane cooled down at liquid nitrogen temperature using a Vitrobot Mark IV (Thermo Fisher Scientific) system. The vitrified samples were then transferred to a 626 cryogenic sample holder (Gatan) and examined with the cryo-TEM at 77 K. Micrographs were captured with a Falcon III camera (Thermo Fisher Scientific).Samples used for SANS measurements were prepared in deuterated water to maximize the scattering contrast between solvent and molecules. Spherical model with hard sphere interference and lamella model were used in the data fitting according the feature of the samples.0n is number density of ions, k is the Boltzmann constant, T is the temperature, h is the film thickness, e is the elementary charge, ψ is the surface potential, and ε is the dielectric constant of the solution. The London–van der Waals contribution was computed using the following expression  calculations were performed with the Gaussian 09 D.01 program package  porphyrin (TPPS) (TPE-diSDS and TPE-SDS atoms restrained by a harmonic potential. Bond lengths were constrained by the LINCS algorithm (All-atom MD simulations were performed with the Gromacs package 4.5.4 (


Morphological and biological properties of the zein nanofiber were determined. Prepared nanofibers were defined by thermogravimetric analysis (TGA), X-ray diffraction (X-RD), Fourier-transform infrared spectroscopy (FT-IR), and scanning electron microscopy. The average fiber diameter was unchanged with an increase in WO3 concentration from 0.001 to 0.008%. FT-IR spectroscopy and X-RD indicated the presence of WO3 in WO3@zein nanofibers. In comparison to WO3-free, WO3@zein nanofibers showed higher safety and preserved the anticancer effect of WO3 against human melanoma cell line (A375) melanoma cells compared to WO3-free. Moreover, both WO3-free and WO3@zein caused a fourfold increase in the cellular proliferation of reactive oxygen species (ROS) in the treated A375 cells compared to untreated cells. ROS elevation led to apoptosis-dependent cell death of A375 cells as evidenced by up-regulating the expression of p53-downstream genes (p21 and Bax) (tumor-suppressor gene) while down-regulating the expression of key oncogenes (BCL2 and cyclin D). In conclusion, the prepared nanofiber represents a promising and safe candidate for anticancer applications.Plant proteins have become attractive for biomedical applications such as wound dressing and drug delivery. In this research, nanofibers from pristine zein (plant protein) and zein loaded with tungsten oxide (WO Predestined annual cancer cases were 18.1 million including 9.6 million deaths-2 years ago- which is expected to double by 20402. Radiation therapy, chemotherapy, and surgery are used as anticancer therapies, but they all have cons such as significant side effects and limited effectiveness3. Cancer issue awareness pushes the scientific community to embark on extensive mechanistic studies and different drug discovery protocols4. The scientific community is currently interested in nanoparticles (NPs) because of their applicable properties and distinctive structural properties5. NPs were used to target cancer tissue precisely due to their small size5. It inducts reactive oxygen species (ROS) that prompt apoptotic cell death in cancer6. ROS play a critical role in both cell cycle progression and apoptosis6. Metal oxides (inorganic NPs) are promising materials for biomedical applications 8. For example, cobalt, iron, and nickel NPs are used in medical biotechnology due to their magnetic properties9. Metal oxide NPs were used as a potential therapeutic against cancer since they caused many effects such as DNA damage, genotoxic effects, induction of oxidative stress, and anti-inflammatory responses10. Tungsten oxide (WO3) NPs is a fascinating transition metal oxide with inclusive applications such as electrochromic, sensors, anti-cancer, and antimicrobial due to its unique structures and properties13. There are various methods to prepare WO3 NPs with diverse morphologies such as nanofibers, nanosheets, one-dimensional nanoneedles, or two-dimensional nanoplates16. NPs can be incorporated into polymer nanofibers via electrospinning polymer solutions containing NPs17. For instance, a facile one-step electrospinning technique was used to construct mesoporous PtO − WO3 nanofibers for highly sensitive and selective acetone sensing17.Cancer is currently recognized as a global problem that unfortunately lacks a worldwide solution20. Nanofibers were prepared by different methods such as drawing techniques, the spinneret-based tunable engineered parameter (STEP) method, phase separation, self-assembly, template synthesis, freeze-drying synthesis, and interfacial polymerization21. The electrospinning technique has recently attracted attention because of its unusual properties22. Notable progress has been made in engineering of electrospun nanofibers and the development of electrospinning techniques to facilitate several applications. Electrospinning produces fibers with tens of nanometer diameters18. Various polymers  are used for preparing nanofibers via electrospinning which have tremendous potential for biomedical applications18. Synthetic polymers are limited for medical applications due to their non-degradable nature, the diversity of chemical composition, or complications owing to their degradation products20. While natural polymers display many vital features, including biocompatibility, biological stability, and biodegradability; thus, they are superior to synthetic polymers21. Zein protein -an example of natural polymers- is categorized as a safe material by food and drug administration (US-FDA)23. Also, it is one of the plant proteins that is used in tissue engineering, food industry, and medical applications24. It is commonly used as a carrier for controlling the release of hydrophobic drugs due to its hydrophobic properties23. Recently, zein microsphere containing quercetin has been expanded as a promising scaffold for tissue engineering25. Zaersabet et al. research has shown that 3D zein scaffold could be a potential candidate for bone tissue engineering due to their promising surface topography, osteoinductivity, biodegradability, and mechanical behavior26. Several herbs and anticancer drugs such as curcumin, exemestane/resveratrol, and daidzin were encapsulated in zein nanofiber29. Although the formation of nanofibers from the huge majority of protein polymers is slightly difficult – because of their complicated macromolecular structure- zein can simply form fibers30. Zein scaffolds were found to have a high porous-walled structure, porosity, microbial attack resistance, adequate biocompatibility, and antioxidant activity31. But, the morphological stability and mechanical weakness of zein fibers in wet conditions are the key limitations. To tackle this issue, it was found that using chemical cross-linkers such as citric acid or glutaraldehyde or incorporating other synthetic polymers enhanced the mechanical properties of prepared zein fibers34. For instance, cross-linked zein fibers (using citric acid) could retain their fibrous structure after submersion in phosphate buffer saline (PBS) for two weeks at 37 C34. Also, addition of polyvinyl alcohol (PVA) and gelatin to zein electrospun (PVA/Zein/Gelatin) has been fabricated to enhance the shelf life of food by assuring the food quality35.In recent years, there has been an incredible surge in the demand for polymeric nanofibers. These remarkable materials hold great promise in a wide range of applications, such as tissue engineering, blood vessels, the nervous system, drug delivery, protective clothing, filtration, and sensors3@zein) that exhibits enhanced characterization suitable for biomedical applications. Nanofibers scaffolds were characterized by Scanning Electron Microscopic Images (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (X-RD), and thermal analysis (TGA). MTT assays on normal human skin melanocyte (HBF4) cell line were performed to assess the cytotoxicity. Finally, the anticancer effect of the WO3@zein nanofiber was evaluated against A375 human melanoma cell line. These nanofibers scaffold could be a promising candidate for treating melanomaIn this study, we employed the electrospinning technique to create polymeric scaffolds using a natural polymer called zein protein. These scaffolds were loaded with Tungsten oxide, resulting in the development of a unique material scaffold (WO36. Human melanoma cell line (A375) was provided kindly from Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications (SRTA-City), New Borg EL-Arab City, 21934, Alexandria, Egypt. All chemicals had high purity and were used without any additional purification.Zein protein was purchased from Sigma-Aldrich . Ethanol (95%) and acetic acid (96%) were purchased from Lonza . Dimethyl sulfoxide (DMSO) was purchased from Merck (Germany). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Lonza (USA), while fetal bovine serum (FBS) was purchased from GIBCO Company (USA). 3--2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Aldrich (Germany). SYBR-green PCR assay kit, cDNA synthesis kit, and Gene JET RNA purification kit were purchased from Thermo Fisher Scientific . Tungsten oxide was provided by our colleague in our institute as it was prepared as earlier reported3@zein), WO3 in amounts of (0.001 and 0.008%) was dissolved in zein solution just before electrospinning to get WO3@zein1 and WO3@zein8, respectively . The nanofibers were collected on a static plate collector at 20 cm from the needle, keeping the feeding rate at 0.25 mL/h. While, WO3@zein solution was electrospun at a voltage of 15 kV, 0.3 mL/h (feeding rate) and 20 cm (collector distance). The nanofibers were collected on sheets of aluminum foil, dried under vacuum overnight to eliminate the residual acetic/ethanol solvent. They kept in a dry/cold condition to prevent any possible contamination. Neat zein and WO3@zein nanofibers scaffold were cross-linked using glutaraldehyde vapor. Table 3 concentration.The zein (40% w/v) was dissolved in acetic acid (96%) and ethanol (95%) in a ratio of 1:1 and stirred for 1 h. To incorporate tungsten oxide in zein nanofiber . Before SEM analysis, the samples were sputtered with gold using a sputter coater . Average fiber diameters were calculated from measurements of 50 fibers from each sample using Image J software .3@zein nanofibers scaffolds. For all spectra, thirty scans were collected from 4000 to 400 cm−1 wavelength with a 4 cm−1 resolution.ATR FT-IR-8400 S  was used to record IR spectra of the WO3@zein nanofibers scaffolds was carried out using Shimadzu X-Ray diffraction 7000 . The radiation wavelength was 1.5406. The data were acquired in the form of 2θ versus intensity (a.u) chart.X-ray diffraction analysis of net zein and WOThermogravimetric Analyzer TGA-50  was used to study thermal stability for prepared nanofibers scaffolds under a nitrogen atmosphere (10 ml/min). Temperature scale was set from 35 to 700 °C with a heating rate of 20 °C/min.3-free and WO3@zein nanofibers scaffolds was evaluated against a normal human skin melanocyte (HBF4) cell line. DMEM medium (supplemented with 10% FBS) was used to culture HBF4 in a 96-well cell culture plate (5 × 103 cells per well) and incubated at 37 ºC in a 5% CO2 incubator. After 24 h for cell attachment, the nanofibers scaffolds WO3@zein as well as WO3-free , were incubated (separately) with HBF4 cells for 72 h (as previously reported37). The cell viability was then evaluated by MTT method38. Effective safe concentration (EC100) of WO3-free and WO3@zein nanofibers scaffolds at which cell viability is 100% was estimated by the Graphpad Instat software. Additionally, morphological alterations of treated HBF4 relative to the untreated cells were captured using a digital camera fitted on a phase contrast inverted microscope .Cytotoxicity of WO3@zein nanofibers scaffolds was evaluated against A375 human melanoma cell line cultured in DMEM (supplemented with 10% FBS) and seeded in sterile 96-well plates (4 × 103 cells/well). After 24 h, the nanofibers scaffolds WO3@Zein as well as WO3-free  were incubated with A375 cells for 72 h at 37ºC in a 5% CO2 incubator. The percentage of growth inhibition of A375 cells was calculated at each corresponding dose, relative to the untreated cells using the MTT method. Moreover, cellular morphological changes before and after treatment with the most effective and safest concentration of WO3@Zein nanofibers scaffolds were captured using a phase contrast inverted microscope with a digital camera .The anticancer effect of the WOThe cellular ROS level was quantified after incubation of the untreated and treated A375 with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 48 h. DCFDA is oxidized by cellular ROS to fluorescent DCF, which was assessed using a spectrofluorometer  at 480 nm excitation and 530 nm emission. The relative fold change in ROS content in the treated cells relative to the untreated was calculated.−ΔΔCT equation .Total RNAs of untreated and the tested anticancer compounds-treated A375 cells were extracted using Gene JET RNA Purification Kit . The cDNA was synthesized from mRNA using cDNA Synthesis Kit . Real-time PCR was performed using SYBR green master mix and specific primers (Forward/Reverse) were mentioned in Table p < 0.05. The unpaired T-tests were used for evaluating the difference between the mean values of the studied treatments. The analysis was done for three measurements using SPSS software version 16.The data are expressed as mean ± standard error of the mean (SEM) and the significant values were considered at 3@zein nanofibers scaffolds that show a ribbon-like morphology with smooth fibers. The average fiber diameter of neat zein nanofiber scaffold (1190 nm) is insignificantly different from WO3@zein1 (1085 nm) and WO3@zein8 nanofiber (1152 nm). Ribbon-like morphology was also reported by Selling et al. when zein was dissolved in aqueous ethanol39. Also, our SEM results agree well with earlier reported results when zein was used as carriers to stabilize (-)-epigallocatechin gallate40. Ribbon-like morphology could be created by the formation of “garden hose” structures that fall to form ribbon-like as they land on the collector41. “Garden hose” structure formed due to the solution droplets (during the electrospinning step) is observed to have an outside skin which collapsed inner as the solvent evaporated41. Morphology and diameter of electrospun zein are affected by several parameters, such as solvent, polymer concentration, distance between collector and spinneret, solution flow rate, and applied DC voltage42. For example, when we used a low concentration of zein (< 40%), beads and fibers were formed and when we used a high concentration (40%), we got continuous fibers. Increasing concentration causes molecular chain entanglement, which prevents the polymer jet from breaking up into droplets40. Also, increasing polymer concentration (to a specific limit) boosts the interaction between the solvent and the polymer, thus reducing the tendency of the polymer solution to form droplets under the effect of solvent surface tension40.Figure 43. Neat-zein displays a considerable peak at 3,289 cm-1 referring to –NH stretching (from amide structure) and an association absorption peak of –OH that is formed by amino acid residues (i.e. adsorbed water on the surface)  and hydrate . Zein is a hydrophobic material and insoluble in water, so surface water acts as a plasticizer for nanofibers. Those results are in harmony with previous results that showed that zein was significantly plasticized by water45. C–H stretching (asymmetric and symmetric) has strong peaks at 2876 cm−1, 2,949 cm−1, and 1446.2 cm−143. Amide III (C–N), amide II (N–H), and amide I (C=O) stretching illustrate peaks at 1245.1 cm−1, 1,532 cm−1, and 1,644 cm−1, respectively46. Those results agree with the previous FT-IR analysis of neat zein44. FT-IR for WO3@Zein nanofiber scaffold shows unchanged peak positions and intensities  and there is no change in HEC peaks37.Proteins have secondary structures due to the presence of amide I and II groups which have distinguished vibrational peaks and so FT-IR provides information about this structure Fig. 43. Neat-3@zein nanofibers scaffolds can be seen in Fig. 47. The first peak describes the α-helix structure of neat zein (intra-helix average backbone distance), while the second peak represents intra-helix packing between neighboring chains49. While, WO3 has two crystalline peaks at 2θ = 23.6°, and 31.8°, may be attributed to (0 0 2) and (2 0 0) planes (Reference pattern ICDD Card No. 00-043-0679) as earlier stated in the literature36. X-RD pattern of WO3@zein nanofiber scaffold shows coexistence of both peaks of WO3 and zein which are broad and less intense, which can be attributed to encapsulation of WO3 and its ability to form amorphous metal50. Also, crystalline peaks of WO3 disappeared in WO3@zein nanofiber scaffold may be due to the covering of crystalline form of WO3 by zein molecules. Those results are a line with Dhanalakshmi et al. when used ZnO Ns with starch molecule to prepare antibacterial film51.X-RD data of neat zein and WO3@zein nanofiber scaffolds are shown in Fig. 3@zein) is examined via thermal gravimetric analysis (TGA) by measuring mass loss as a function of temperature52. In all samples, the TGA curve has two weight loss stages. First stage (44.5–66.9 °C) refers to the loss of water and any residual solvent (acetic acid: ethanol) from the nanofiber53. Second stage (213–456 °C) relates to the progressive depolymerization and decarboxylation of thermally unstable protein units54. These results are in agreement with those obtained in previous TGA55. Furthermore, WO3 addition does not affect zein thermal stability due to the low concentration of WO3 compared to zein concentration. These results are consistent with those found when WO3 (low concentration) was added to hydroxyethyl cellulose (HEC) and HEC thermal stability remained unchanged37.Thermogravimetric curves of neat zein and WO3-free and WO3@zein nanofibers scaffolds is tested against HBF4 normal melanocytes and A375 cells to determine its potential toxicity on normal cells  reduce to 63.09%, 53.41%, and 33.86%, respectively, compared to 91.64%, 85.68, and 76.14%, respectively, in the case of WO3@zein nanofiber scaffold. Therefore, the growth of WO3@zein-treated HBF4 cells is maintained at > 76% even at the highest dose (8.6 mM). These results confirm the ability of the newly developed formula to reduce the toxic effect of WO3 on normal cells. Tungsten was reported to accumulate in several organs and/or tissues such as kidneys, liver, ovaries, and uterus causing detrimental toxic effects which have limited its biomedical applications56. Limitations of metal/metal oxide toxicity, instability, and aggregation can be overcome through incorporation or encapsulation into polymers58. Polymers deliver metal/metal oxide with lower toxicity but with augmented efficiency when given at reduced doses 59. For instance, the cytotoxicity of WO3  was decreased when incorporated into hydroxyethylcellulose 37.Cytotoxicity of WO3-free and WO3@zein nanofibers scaffolds at the same concentrations that were used in the cytotoxicity test. Results show that the nanofibers scaffolds sustain the growth inhibitory effect of WO3@zein nanofiber scaffolds on A375 melanoma cells  compared to the WO3-free form  at doses of 0.3, 4.3, and 8.6 mM, respectively. Also, the figure shows comparable damage in both normal and melanoma cell lines after 72 h treatment with WO3-free (0.3 mM). Both melanoma and normal cells are inhibited by 50.86% and 36.91%, respectively. In contrast, WO3@zein nanofiber scaffold (at the same concentration) exhibits only morphological damage in treated melanoma cells (which causes 50% growth inhibition) without having a significant effect on normal cell proliferation. The concentration of 0.3 mM of WO3 has been selected for further evaluation of the anticancer potential of the WO3@zein nanofiber scaffold. The pro-oxidant activity of tungsten oxide has been suggested that tungsten-containing compounds can act as catalysts for oxidation reactions60. Several studies have confirmed that tungsten oxide can elevate intracellular reactive oxygen species (ROS) and reduce the antioxidant capacity of mammalian cells61.Figure 3-free and WO3@zein nanofiber scaffold (0.3 mM concentrations) in comparison to untreated control cells  relative to untreated cells. ROS are biomolecules with multiple functions in mammalian cells related to cellular signaling. Under normal cell conditions, elevated ROS levels induce oxidative stress, leading to cellular senescence, carcinogenesis, or cell death. Therefore, sustaining redox homeostasis in normal cells is crucial for cell survival62. On the contrary, metabolically demanding cancer cells have an elevated level of ROS due to uncontrolled proliferation63. However, a massive accumulation of ROS in cancer cells inhibits tumor growth by suppressing the proliferation signaling pathway, cell cycle, and the biosynthesis of nucleotides and ATP64. Therefore, finding pro-oxidant agents able to deprive cancer cells of the ability to control ROS levels is critical.Reactive oxygen species (ROS) content of A375 cancer cells is evaluated after treatment with WO3 (WO3-free and WO3@zein nanofiber scaffold) using qPCR. Oncogenes (B-cell lymphoma 2 “BcL-2” and cyclin D-mediated uncontrolled cell cycle) and for up-regulation of proapoptotic genes (cyclin-dependent kinase inhibitor p21 and Bcl-2-associated X protein “BAX”) are used as examples65. Treatment with WO3 (WO3-free and WO3@zein nanofiber scaffold) induced upregulation of p53-downstream genes (p21 and Bax) while downregulating oncogenes (BCL2 and cyclin D). Pro-oxidant activity of WO3-free and WO3@zein nanofiber scaffold can be linked to its suppressive effect on BCL2 and cyclin D through many explanations. For instance, Hildeman et al. reported that ROS could sensitize T cells to apoptosis by decreasing the expression of Bcl-266. Similarly, BCL-2 inhibition in MIN6 mouse insuloma cells increased the production of peroxides67. Besides, exposure of HeLa human cervical cancer cells and HEK293 human embryo kidney cells to excessive ROS induced inhibition of cyclin D1. This was found to contribute to the induction of cell cycle arrest in G2 phase under oxidative stress68. The pro-oxidant activity of WO3-free and WO3@zein nanofibers scaffold is also linked to its promoting effect on the apoptosis-related genes p21 and Bax. For example, induction of apoptosis by p21 in sarcoma cell lines can be ameliorated with antioxidants and sarcoma cells undergoing p21-dependent cell death had higher sensitivity to oxidants69. Thioridazine induces Bax-dependent apoptosis by enhancing ROS production followed by ER stress70.Figure 3) as a metal oxide was incorporated into zein nanofibers scaffold using electrospinning technique. Morphology and the thermal stability of zein nanofibers scaffold unchanged by WO3 addition. While, FT-IR and X-ray diffraction (X-RD) data confirmed the presence of WO3 in WO3@zein nanofiber scaffold. Cytotoxicity of WO3@zein nanofibers scaffolds confirmed its ability to reduce the toxic effect of WO3 on normal cells as the growth of HBF4 cells was maintained at > 76% even at the highest dose (8.6 mM). Moreover, anticancer results for WO3@zein nanofibers scaffolds exhibits only morphological damage in treated melanoma cells (which causes 50% growth inhibition) without having a significant effect on normal cell proliferation. The results demonstrate that WO3@zein nanofiber scaffold prepared by electrospinning could be used as a promising material candidate as anti-melanoma.In this paper, we have described an effective process to prepare biocompatible nanofiber scaffold from plant protein (zein) as anti-melanoma. Tungsten oxide (WO


Self-focused attention (SFA) is a major maintenance factor of social anxiety disorder. The two types of SFA, the observer perspective and self-focus on body sensation, increase anxiety in individuals with high levels of social anxiety. However, the triggers of each SFA remain unclear. This study used ecological momentary assessment to identify the factors that elicit SFA in real-life social scenarios.The study obtained 316 samples from 22 Japanese university students  with high social anxiety who completed momentary measures of stimulus perception and two types of SFA for 10 days. Links to online questionnaires were sent to the participants via e-mails 3 times a day. First, multilevel single regression analyses were used to identify the stimuli that induced the two types of SFA. Between-level interaction with gender was done to determine the effect of gender biasing on the female participants. Next, for the variables that were significantly predictive in these analyses, multilevel multiple regression analyses were conducted with fear of each stimulus as a control variable.Perception of gaze, evaluation, and authority predicted SFA from the observer perspective. Perception of gaze also predicted self-focus on body sensation. In addition, the perception of positive response and that of stranger predicted self-focus on body sensation depended on gender, implying that the positive response perception of female participants predicted self-focus on body sensation. After controlling for corresponding fear, gaze perception predicted both SFAs, and the perception of authority predicted SFA from the observer perspective. In addition, after controlling for relevant fear, the perception of positive response of female participants predicted self-focus on body sensation. In contrast, the fear of evaluation but not the perception of evaluation predicted SFA from the observer perspective.The perception of gaze is the most powerful trigger of the two types of SFA, even after controlling for fear of gaze in real-life social scenarios for individuals with social anxiety. SFA from the observer perspective is also triggered by the perception of authority and fear of evaluation. The role of perception of positive responses or strangers should be re-evaluated after correcting for gender imbalance. (350 words / 350 words) Social anxiety disorder (SAD) is a psychiatric disorder characterized by marked fear or anxiety regarding social situations in which an individual is exposed to scrutiny from others . SAD disSelf-focus on body sensation is also emphasized in cognitive-behavioral models of SAD . For exaThe cognitive-behavioral model of Clark and Wells  suggestsIn addition to the affective valences of audience behavior, SFA may have some other triggers in a social situation. For example, in a speech task with a social evaluative threat, individuals with high levels of speech anxiety displayed high SFA levels compared with those with low levels of speech anxiety . SpecifiPrevious studies on SFA utilized traditional methods with one-time measures using laboratory designs, such as speech tasks or recalling specific social situations, whereas only a few captured natural social scenarios. The current study used ecological momentary assessment (EMA) to identify the triggers of SFA in multiple natural social situations. EMA is an ecologically valid method that uses a repeated collection of real-time data on the behavior and experience of subjects in their natural environment . ConventThe current study had two objectives. The first was to identify the factors that elicit each of the two types of SFA in real-life social situations through the use of EMA. We focused on the perception of various stimuli in social situations as triggers of SFA. We investigated whether SFA would be elicited by perceiving each of nine stimuli: gaze, negative response, positive response, evaluation from other people, speaking environment, and the presence of authority, a stranger, an acquaintance, and a familiar person. The second objective was to identify the triggers that increase SFA according to the perception per se in a social situation. For example, because fear of evaluation is associated with SFA, fear of other stimuli may also be related to SFA by comprising the internal cues of those stimuli. Thus, fear of the nine stimuli was measured as a control variable, where the effect of fear was controlled.SD = 23.37, minimum = 35, and maximum = 128), whereas the average age was 20.82 years (SD = 2.06). All participants were Japanese and reported no psychological disorders, including SAD or traumatic experiences. After the survey, we gave the participants a book voucher worth 1000 JPY.The participants were recruited from a single university, the one the authors belong to, through an advertisement on a portal site available to all university students. They were screened for social anxiety using the Japanese version of the Liebowitz Social Anxiety Scale (LSAS-J) , and thenot at all; 3 = totally) to assess fear and avoidance of 24 common social performance and interaction situations. Scores range from 0 to 144, where the sum of each item is used to rate the severity of social anxiety. The cut-off point of the LSAS-J is 44, whereas the boundary value is 30. The measure has a high internal consistency (α = 0.95) and test-retest reliability (0.92). The LSAS-J has been correlated with the Japanese version of the Social Avoidance and Distress scale  [As previously mentioned, the LSAS-J was used to screen for social anxiety. The LSAS-J uses a four-point Likert-type scale (0 =  < .001) .Table Social situation. This question was used to ensure whether the participants experienced certain social situations. The scale included nine representative social situations extracted from the items of the LSAS-J. Four social situations denote interactional communication, such as “participating in small groups or meetings” and “having a one-on-one conversation.” Another four situations pertain to performance, in which people act in front of others, such as “acting in front of an audience” and “working while being observed.” In addition, being in a crowded place is included in the nine social situations. These situations were not only in-person but also online, such as through video conferences. The participants selected one situation that they experienced within 5 hours before answering. They selected the option “I did not experience any social situations” when they had not experienced any of the social situations. After selecting one of the social situations, they were requested to describe the start and end times, the place, and the people with them.Perception of stimuli in a social situation. This question assessed whether the participants perceived nine stimuli  in the social situations using a two-point scale (0 = no or 1 = yes).Fear of stimuli. This question uses a six-point Likert-type scale  to rate nine items and measures of the fear of each stimulus in the social situations.SFA from the observer perspective. This question measures the degree of SFA from the observer perspective in the situation. The items are derived from the Mental Perspective Scale for Social Anxiety Disorder (MPS) [Strongly disagree; 6 = Strongly agree).er (MPS) . The MPSer (MPS) . We usedSelf-focus on body sensation. This question was modified from the Focused Attention Scale (FAS) [Strongly disagree; 6 = Strongly agree).le (FAS) . The FASle (FAS) . The FASle (FAS) . The parAt the first online meeting, the participants were instructed to respond to the questionnaire using smartphones for 10 days. They were sent e-mails with a link to the questionnaire three times per day during the EMA survey: 12:00, 17:00, and 22:00. Five-hour intervals were used to reduce the burden on participants because some social situations in daily life, such as classes or part-time jobs, may last a long time and participants may experience other social situations, such as team meetings, less frequently in a day. The participants were told to respond to the questionnaires within one hour of receiving the e-mail. If an hour passed without their response, they were asked to ignore the prompt and answer the questionnaire in the following e-mail. The questions were related to social situations that the participants experienced within 5 hours before answering. They were instructed that interaction with family members was excluded from social situations, whereas online situations, such as video conferences, were included. They received compensation after the EMA survey.The EMA data were analyzed through the use of HAD (ver16.03) . The datWe sent the participants 660 questionnaires in total, of which 109 were unanswered. Thus, 551 responses were collected in the present EMA survey, and the compliance rate for EMA input was 83.5%. Responses that indicated that the participants experienced no social situations , responses describing interaction with family members (3 responses) and responses related to experiences that occurred more than 5 hours before the answer (5 responses), were excluded from the analysis. The final sample included 316 responses.r =. 45, p < .01).Table The center three columns of Table r = .52, p < .010), between positive response and speaking environment , and between evaluation and authority . A significant, moderate, negative correlation was shown between stranger and familiar person .Table b (293) = 2.10, p < .001; evaluation: b (293) = 1.67, p = .001; authority: b (293) = 2.04, p = .003). In addition, gaze significantly predicted self-focus on body sensation (b (293) = 1.60, p < .001).Table p-value of 0.0056 was used. The results showed that the interaction between a positive response and gender was significant in the model with self-focus on body sensation as the dependent variable (b (292) = -2.27, p < .001), although the main effect of a positive response was not significant. A simple slope analysis showed that the main effect was significant for both the female and male groups  = 0.91, p = .001, male: b (292) = -1.36, p < .001), which means SFA was increased in the female group by perceiving positive response, and vice versa was found for the male group. The interaction between stranger and gender was also significant in the model with self-focus on body sensation as the dependent variable (b (292) = 3.04, p = .004), although the main effect of stranger was not significant. A simple slope analysis showed that the main effect was marginally significant for the male group (b (292) = 2.59, p = .006). The interaction between authority and gender showed a marginally significant p-value of 0.008 (b (292) = -3.09). A simple slope analysis showed that the main effect was significant in the female group (b (292) = 2.49, p < .001).To investigate the effect of gender, we included it in the interaction term as a level 2 variable in the analyses examining the relation between stimuli perception and SFA. Bonferroni with a corrected r = .20, p < .010; evaluation and fear of evaluation: r = .34, p < .010; and authority and fear of authority: r = .23, p < .010). In addition, positive response and stranger, which had significant interactions with gender, were examined for the correlation with their fearful affect. They both displayed low significant correlations with their corresponding feelings of fear . Thus, we regarded multicollinearity as not being a problem for the multiple regression analyses.Multilevel correlation analyses between the stimuli that significantly predicted SFA, shown in Table b (292) = 1.47, p < .001). Second, the results for evaluation demonstrated that it did not predict SFA (b (292) = 0.68, p = .074), while fear of evaluation did predict SFA (b (294) = 0.90, p < .001). Third, the results for authority demonstrated that it significantly predicted SFA from the observer perspective after controlling for relevant fear (b (292) = 1.83, p = .004). Fourth, gaze was analyzed as the independent variable, self-focus on body sensation was designated as the dependent variable, and related fear was used as the control variable. The results illustrated that gaze perception significantly predicted self-focus on body sensation (b (292) = 1.30, p = .001).The upper three variables of Table p-value was 0.017. The interaction between gender and positive response was significant (b (292) = -2.18, p < .001), while the main effect of positive response was not. A simple slope analysis showed that the main effect was significant for both the female and male groups  = 0.85, p = .002, male: b (292) = -1.33, p < .001), which means SFA increased in the female group by perceiving positive response, and vice versa was seen in the male group. The interaction between gender and stranger was significant (b (292) = 3.02, p = .007), while the main effect of stranger was not. A simple slope analysis showed that the main effect was significant for the male group (b (292) = 2.48, p = .015). These results were almost the same as shown in the analyses without controlling for corresponding feelings of fear.The lower two variables of Table This study used EMA to capture the perception of stimuli and two types of SFA in real-life social situations. Individuals with high levels of social anxiety displayed increased SFA from the observer perspective when perceiving gaze, evaluation, and authority in everyday social situations. They also displayed increased self-focus on body sensation when perceiving gaze. In addition, for positive response and stranger, there was a gender effect in predicting self-focus on body sensation. Considering the scarcity of male participants, the significant result for positive response and the marginally significant result for stranger in the male group should be interpreted cautiously. Still, it can be said that the female group displayed increased self-focus on body sensation when perceiving positive responses. The perception of gaze increased both types of SFA, even when the perception of stimuli was controlled for fear of gaze. Thus, the perception of gaze would be a strong candidate for the specific aspect of social situations necessary to trigger an internal shift of attention reported by Schultz and Heimberg .The observer perspective should be triggered by perceiving the presence of an audience . TherefoIn the current study, the perception of positive response predicted self-focus on body sensation in the female participants, which was maintained even after controlling for corresponding fear. In addition, the perception of evaluation did not predict self-focus on body sensation, which means that the perception of positive response per se, not related to fear or being positively evaluated, affected the abovementioned result. Socially anxious individuals tend to interpret positive social events in a way that maintains a sense of social threat . FurtherThe finding that the perception of negative response from others did not predict either type of SFA was inconsistent with the models of Clark and Wells  and HeimThe speaking environment did not significantly predict either type of SFA. This factor differed from others in that it may indicate not the perception of but participation in such an environment. Although it did not significantly predict either type of SFA, the coefficient was greater for SFA from the observer perspective than for self-focus on body sensation. This may denote a difference between the two types of SFA. The perceptions of acquaintance and familiar person were also not relevant to either SFA. The results suggest that closeness to the interaction partner is not associated with SFA. In the current study, SFA may be related to whether the interaction partner is higher in social status, such as in a position of authority, than oneself in the situation rather than whether the partner is close or not.Results related to the second objective indicated that only the perception of gaze increased both types of SFA after controlling for relevant fear. Patients with SAD exhibit abnormal gaze perception compared with control subjects . Using “The perception of evaluation did not predict SFA from the observer perspective after controlling for its fear, but that fear significantly predicted the SFA. Thus, it was not the evaluation accompanying negative and positive responses that predicted SFA from the observer perspective, but it was the fear of that evaluation that predicted the SFA. Both the fear of negative evaluation and that of positive evaluation are claimed as strong predictors of SAD , 40, andThe current study has several limitations, and we suggest future directions to address these limitations. First, although the time periods were relatively short, there is still the possibility of recall bias. The participants had to recall a situation they had experienced a few hours previously, and the process of recalling a social situation might distort or generalize somewhat the content of memory. Therefore, future studies should be designed to have participants report during or immediately after experiencing social situations to minimize recall bias. Second, this study used only subjective reports. We could not objectively measure the details of the actual social situation because the present EMA study measured variables according to participant responses through smartphones. As previously mentioned, patients with SAD exhibit abnormal gaze perception. Thus, the participants may have responded that they perceived gaze from others even when no one gazed at them in social situations. In addition, the other stimuli might also be overreported or underreported. Thus, future studies should manipulate stimuli, including gaze from others in a social situation, and should investigate the relation between the presence of stimuli, subjective perception, and SFA. Liao et al.  developeThis was the first study to use EMA to explore the perception of a wide variety of real-life social situations eliciting SFA. The results demonstrated that the perception of gaze was a common trigger for SFA, both from the observer perspective and body sensation, while the perception of evaluation and authority were also triggers for SFA from the observer perspective. The perception of positive response was also a trigger for the self-focus on body sensations of female participants. Furthermore, gaze perception was relevant to both types after controlling for fear of gaze.