Previous evidence links the formation of extranuclear inclusions of transcription factors, such as ERK, Jun, TDP-43, and REST, with oxidative, endoplasmic-reticulum, proteasomal, and osmotic stress. To further characterize its extranuclear location, we performed a high-content screening based on confocal microscopy and automatized image analyses of an epithelial cell culture treated with hydrogen peroxide, thapsigargin, epoxomicin, or sorbitol at different concentrations and times to recreate the stresses mentioned above. We also performed a subcellular fractionation of the brain from transgenic mice overexpressing the Q331K-mutated TARDBP, and we analyzed the REST-regulated mRNAs. The results show that these nuclear proteins exhibit a mitochondrial location, together with significant nuclear/extranuclear ratio changes, in a protein and stress-specific manner. The presence of these proteins in enriched mitochondrial fractions in vivo confirmed the results of the image analyses. TDP-43 aggregation was associated with alterations in the mRNA levels of the REST target genes involved in calcium homeostasis, apoptosis, and metabolism. In conclusion, cell stress increased the mitochondrial translocation of nuclear proteins, increasing the chance of proteostasis alterations. Furthermore, TDP-43 aggregation impacts REST target genes, disclosing an exciting interaction between these two transcription factors in neurodegenerative processes. The presence of protein aggregates is a pathological hallmark for several neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease, and amyotrophic lateral sclerosis (ALS), to name a few . These pIn the case of ALS, TDP-43 is a primary component of these aggregates. TDP-43 is a ubiquitous protein that belongs to the heterogeneous nuclear ribonucleoprotein family and is encoded by the TARDBP gene. TDP-43 is mainly found in the nucleus of normal cells and is involved in RNA regulation, including transcriptional regulation, alternative splicing, and mRNA stabilization . TDP-43 In addition to TDP-43, other proteins with roles as transcriptional factors also show their cytoplasmic accumulation as aggregates . We inclThe dysfunction of the nuclear pore complex is linked to cytoplasmic mislocalization and TDP-43 aggregation, in addition to the dysregulation of stress granule formation . Indeed,Additionally, p-TDP-43, p-ERK , and RES2O2 in HMEC cells led to changes in the nuclear and non-nuclear distribution of p-TDP-43, while the nuclear intensity of p-TDP-43 was decreased in the milder oxidative conditions tested , followed by sex , but not the transgenesis , with sex interacting with the subcellular location also being a relevant factor .Concerning ERK, the findings in the spinal cord of that murine model reinforce the in vitro data. Thus, in line with the colocalization of p-ERK with complex V, the crude mitochondria showed a relevant concentration of p-ERK. However, the nuclear fractions showed a high concentration of p-ERK, while the total ERK levels were significantly lower in the nuclei than cytosol . Densitop < 0.0001), with sex also influencing the values . TDP-43 overexpression only explained 4% of the total variance in the sex and subcellular location  and sorbitol incubation  strongly influenced the TDP-43 immunostaining intensity. In this particular model, we explored the effect of REST targets on SHSY-5Y cells. The results of the RT-qPCR revealed that TDP-43 aggregation induced by sorbitol incubation was associated with significant decreases in the mRNA levels of SCN3B, FADD, PUMA, DAXX, SOD1, CAT, GAP43, 1433, MAPK11, MAPK12, and ARC, known REST targets. Sorbitol incubation also increased some REST targets such as NRX3 and ATP2B2 . Thus, after sorbitol incubation C, both td ATP2B2 D.To evaluate if TDP-43 aggregation can be related to mitochondrial dysfunction, we evaluated if sorbitol incubation in these conditions led to alterations in the cellular ATP production. The results  suggest p < 0001).We also examined if other ALS-related cell stressors, such as ER stress, could induce similar delocalizations of the evaluated proteins. We have previously shown that proteasomal and ER stress induce a cytosolic mislocalization of TDP-43 . Using sp < 0.0001). In p-Jun, there was a significant interaction between stress and the subcellular location, i.e., the effect on epoxomicin depended on the location. Therefore, epoxomicin treatment decreased the cytosolic levels of p-Jun  increase in the REST intensity in the cytosol was followed by a decrease in the nuclear amount at 4 h, A. The deThis work shows that several proteins implicated in ALS  and other neurodegenerative processes, such as AD , show a sensitivity to cell stress. Several neurodegenerative diseases exhibit protein aggregates where these proteins may be present . We evalIn the case of TDP-43, the results showed that, after oxidative stress or osmotic stress, the cytosolic levels of this protein increased both in the endothelial cell line and in the neuronal cell lines. Further, the degree of colocalization within the mitochondria also increased. Of note, TDP-43 may show differences in behavior with p-TDP-43. Thus, while TPD-43 is accumulated after sorbitol stress in the cytosol, it was nonsignificant in p-TDP-43. The in vivo evidence presented shows that this is also the case in the brain lysates, where p-TDP-43 and TDP-43 show differential responses to the overexpression of mutated TARDBP. Whatever the case, the subcellular fractionation data agree with the enrichment of both endogenous murine tdp-43 and hTDP-43 in mitochondrially enriched fractions. Both the functional and morphological analyses showed that the conditions linked to p-TDP-43 aggregation  were associated with major changes in the mitochondrial ATP production. We should indicate that sorbitol incubation is quite harsh, which could impair the mitochondrial function by several pathways.Regarding ERK, in response to increased oxidative stress, we noticed a decrease in the cytosolic and nuclear locations after a transient increase. Nonetheless, the degree of colocalization increased significantly, suggesting the close occurrence of p-ERK with mitochondrial epitopes again. In contrast with p-TDP-43, the slopes relating to nuclear and extranuclear p-ERK were inversely related to the oxidative stress intensity, suggesting the nuclear retention of this factor or rapid cytosolic clearance of it. The same phenomena (decreased both in the cytosol and in nuclei) was present after proteasome and ER stress. This later cell stress also decreased the colocalization within the mitochondrial epitopes. Interestingly, after the epoxomicin treatment, we observed an increase in the degree of mitochondrial colocalization. The subcellular fractionation in the TDP-43 model suggested that p-ERK could be significantly enriched with mitochondria, independently of the TARDBP overexpression.In the present work we show, concerning p-Jun, no clear accumulation in the extranuclear location was present after oxidative stress, though the mitochondrial colocalization increased. After proteasome inhibition and ER stress, the extranuclear levels of this factor were decreased with concomitant retention in the nuclei . In clear contrast with oxidative stress, both ER stress and proteasome inhibition decreased the mitochondrial colocalization. Indeed, the subcellular fractionation suggested that, while Jun accumulated with crude mitochondria, this was not the case with p-Jun.Regarding REST, its accumulation has been previously reported in a cytosolic location in neurodegenerative processes. Our data regarding the response to cell stress showed that its behavior was similar to p-Jun, showing an initial decrease in the cytosol followed by a slight increase in both the nuclei and cytosol. Epoxomicin treatment decreased the nuclear levels after the long exposure, while ER stress increased their values in the cytosol and nuclei. Indeed, the degree of colocalization increased after oxidative stress. Previous data also reported the relationship between changes in the REST expression and protein aggregates . Particu++ pump. Additionally, we noticed a decreased ARC mRNA expression in the cells, implicating altered mRNA traffic (one of the functions of TDP-43). We also detected an increased NRXN3 expression, encoding neurexin 3. This factor encodes a cell adhesion component whose paralogs have been recently implicated in synaptic strength, in close collaboration with the factors regulating RNA/aggregation toxicity [Interestingly, the in vivo data showed that the overexpression of mutated TARDBP increased the degree of REST enrichment in mitochondrial fractions. The interaction between TDP-43 alterations and REST is new, and it is reinforced by the fact that several REST-regulated genes appear affected under osmotic stress conditions, where TDP-43 is mislocalized. These genes included SCN3B, encoding a voltage-gated sodium channel , and twotoxicity . We alsotoxicity . This butoxicity . NoteworFoxo 1 mRNA. It is known that this key metabolic transcription factor promotes neuron death [We also detected a tendency for increased on death . TherefoAccounting for the fact that one of their primary functions is working as transcription factors  or interacting with RNA (in the case of TDP-43), their localization in the extranuclear placements  suggests the existence of pathogenic mechanisms operating in common. All these factors require nucleocytosolic transport; therefore, a loss in this cellular property’s homeostasis may partially explain their presence in the cytosol. Indeed, we have recently reported that the nuclear envelope (where the proteins responsible for nucleocytoplasmic shuttling reside) showed altered properties in ALS patients and models .Several reports have described the occurrence of TDP-43 in mitochondrial fractions ,28,29 anRegarding p-ERK, it is known that there are mitochondrial substrates for its kinase activity . Recent To the best of our knowledge, we have not found previous evidence of REST in the mitochondria. Previous evidence in human substantia nigra  showed aAs for the limitations of our work, we must remark that this evidence was present in an endothelial-like cell phenotype. Therefore, neuronal or glial cells could exhibit different dynamics. Nonetheless, some of the findings in the endothelial cell culture were replicated independently in lysates of the brain cortex of a murine model, and in N2A and SHSY-5Y cells, human neuronal lines further validated the potential findings. We shall also indicate that the subcellular fractionation was more enriching than a strict purification of the indicated compartment. However, we can exclude nuclear contamination of the mitochondrial fraction (based on purity markers).Cell stress related with ALS enhances the non-nuclear localization of transcription factors. These changes could be modeled in vitro with several cell types and may be related to impairments in nucleocytosolic traffic. These transcription factors, under cell stress, increase their interactions with the mitochondria and potentially influence their physiology (as shown for the TDP-43-REST interaction). In particular, TDP-43 aggregation was associated with alterations in the mRNA levels of the REST target genes involved in calcium homeostasis, apoptosis, and metabolism.n = 5 different mice from each sex) and from 90 days old, an age where the motor phenotype is not present. Housing and obtention of animals were as described [According to local laws and the Directive 2010/63/EU of the European Parliament, all experimental procedures were approved by the Institutional Animal Care Committee of the University of Lleida. The minimal number of animals was calculated according to the deviation of western-blot profiles in previous experiments . Both noescribed ,44. For g to obtain a nuclei-enriched fraction. The supernatant was washed twice by centrifuging for 10 min at 1500× g to eliminate any residual whole cells and cell debris. Supernatant obtained was centrifuged for 15 min at 10,000× g; the supernatant contained the ER/cytosol fraction, and the pellet contained the crude mitochondrial fraction. The ER/cytosol fraction was centrifuged for 1 h at 100,000× g in an ultracentrifuge and the supernatant was considered cytosol.Purification of nuclear, mitochondria and cytosol enriched fractions performed as described . Briefly2.Human mammary epithelial cells (HMEC) cell line  was grown in DMEM medium  supplemented with 10% fetal bovine serum heat inactivated (Invitrogen), 2-mM L-Glutamine (Invitrogen) and 20-U/mL penicillin and 20-µg/mL streptomycin (Invitrogen) as antibiotics. The cells were kept at 37 °C in humidify atmosphere with 5% of CO2.N2A and SHSY-5Y (ATCC) cell lines were grown in Advanced MEM medium (Invitrogen) supplemented with 10% fetal bovine serum heat inactivated (Invitrogen), 2-mM L-Glutamine (Invitrogen) and 20-U/mL penicillin and 20-µg/mL streptomycin (Invitrogen) as antibiotics. The cells were kept at 37 °C in humidify atmosphere with 5% of CO2O2 , 5-µM thapsigargin (Thp) (Millipore-Sigma), and 2.5-µM epoxomicin (Epox) (Millipore-Sigma), respectively, for 2 or 4 h or sorbitol (Millipore-Sigma) 0.4 M for 3 h. To avoid the influence of growth factors present in the fetal bovine serum on the results, the normal culture medium was replaced by Opti-MemTM (#31985062 Invitrogen) before 12 h before all assays, as described [To study the effects of oxidative stress, alteration of endoplasmic reticulum, inhibition of proteasome activity and osmotic stress, cells were treated with 10-µM Hescribed . Cell viTM, Invitrogen, Waltham, MA, USA) for 12 h were treated as indicated above. After incubation, cells were washed with PBS and then fixed with 3.7% paraformaldehyde for 10 min at room temperature. Cells were rinsed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 30 min and subsequently blocked with 5% normal goat serum at room temperature for 1 h. Cells were incubated with (1) the mouse anti-p-TDP-43 (pS409/410) monoclonal antibody (TIP-PTD-M01) ; (3) the rabbit anti-p-ERK 1/2 polyclonal antibody (4370) ; the rabbit anti-REST polyclonal antibody (ab21635) ; (4) the rabbit anti p-Jun polyclonal antibody ; and (5) the anti-ATP5A mouse monoclonal antibody (ab14748)  at 4 °C overnight. After 3 washes with 0.1% Triton X-100-PBS at RT for 10 min, cells were incubated with Alexa Fluor-488 goat anti-rabbit IgG or Alexa-Fluor-594 goat anti-mouse IgG -conjugated secondary antibody. Nuclei were stained with 4′,6′-diamino-2-phenylindole (DAPI) . The coverslips were mounted in Fluoromount-G (Southern Biotech) and images were taken with an Olympus FV10i laser scanning confocal microscope and an Olympus FV1000 confocal microscopy, 60× magnification. For the evaluation of nuclear and cytosolic protein intensity, we used a dedicated pipeline created on the open source CellProfiler software [Cells, seeded out on coverslips and incubated in serum and phenol red free medium . Gels were blotted onto PVDF membranes by transfer at a constant 100 volts, 1 h at RT. The membranes were then blocked by nonfat dry milk solution (5%) in 1 × TBS  and incubated in the desired primary antibody overnight.Protein homogenates were prepared in the presence of inhibitors of phosphatases (Sigma-Aldrich) and proteases . Protein concentration of the samples was measured using the Quick Start™ Bradford 1× Dye Reagent (Bio-Rad #5000205). Proteins were detected by immunoblotting using horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (Santa Cruz Biotechnology). Protein samples were run on SurePAGEMembranes were then washed in 1 × TBST with Tween 20 (0.05%) three times, 5 min each before incubating with the secondary antibody for 1 h at room temperature. Following the secondary antibody incubation, membranes were washed in 1 × TBST with Tween 20 (0.05%) three times, 5 min each and one time, 5 min, with TBS 1 ×. Blots were imaged in the Chemidoc MP Imaging System following incubation in the Immobilon ECL Ultra Western HRP Substrate .TM Precast gels . Cruz Marker™ molecular weight standards  were loaded in the gels. Gels were blotted onto low fluorescence PVDF membranes (Immobilon®-FL PVDF: sc-516541). Nonspecific binding was blocked in incubating membranes with UltraCruz® Blocking Reagent (sc-516214) for 1 h at room temperature, with shaking.For Jun/ p-c-Jun Westerns, protein samples were run on SurePAGE® conjugated primary antibodies (Anti-p-c-Jun Antibody (KM-1) Alexa Fluor® 790 and Anti c-Jun Antibody (G-4) Alexa Fluor® 680, from Santa Cruz) diluted 1:1000 in UltraCruz® Blocking Reagent. Cruz Marker™ MW Tag-Alexa Fluor® 680 (sc-516730) and Cruz Marker™ MW Tag-Alexa Fluor® 790 (sc-516731) at 1:1000 were added to obtain molecular weight distribution.The blocked membranes were incubated with the appropriates Alexa FluorMembranes were incubated in this mixture for 2 h at room temperature, in the dark, with shaking. Membranes were then washed three times for 5 min each with TBST and once for 5 min with TBS. Blots when then placed on the top of blotter paper and dried for 5–10 min. The western blot was imaged using the infrared (IR) laser-based instrumentation LI-COR Odyssey .RNA was extracted from cells and brain lysates using TRI Reagent  following the manufacturer’s instructions. RNA concentrations were measured using a NanoDrop ND-1000 (Thermo Fisher Scientific). One microgram of RNA was used for retrotranscription to cDNA employing TaqMan Reverse Transcription Reagent and random hexamers .RT-qPCR experiments were performed using a CFX96 instrument  with SYBR Select Master mix for CFX . Each 20 µL reaction mix contained 4-µL cDNA, 10-µL SYBR Select Master Mix, 0.2 nM of forward primer and 0.2 nM of reverse primer solutions and 4-µL PCR grade water. RT-qPCR run protocol was as follows: 50 °C for 2 min and 95 °C for 2 min, with the 95 °C for 15 s and 60 °C for 1 min steps repeated for 40 cycles, and a melting curve test from 65°C to 95 °C at a 0.1 °C/s measuring rate. Primers employed in these experiments, previously described in reference [Mitochondrial function was estimated employing the Seahorse XF HS Mini Analyzer (Agilent technologies). Briefly, to obtain an effect of the aggregation-prone conditions in the mitochondrial function we employed the Seahorse XFp Real-Time ATP Rate assay kit (Agilent#103591-100), according to the manufacturer’s instructions. 20,000 SHSY-5Y cells were plated in the microplates of the kit and incubated with sorbitol . After this incubation, oxygen consumption and extracellular acidification, were recorded, in basal conditions, and after oligomycin and rotenone/antimycin addition.t-tests, One-way, Two-way, and Three-way ANOVA analyses, with adequate post-hoc analyses, once normality of variables was tested by Kolmogorov–Smirnov test. The 0.05 level was selected as the point of minimal statistical significance in every comparison.All statistics were performed using the GraphPad Prism version9.1.2 for Windows software . Differences between groups were analyzed by the Student’s


Reactive oxygen species (ROS) encompasses a collection of complicated chemical entities characterized by individually specific biological reactivities and physicochemical properties. ROS detection is attracting tremendous attention. The reaction-based nanomaterials for ROS “turn-on” sensing represent novel and efficient tools for ROS detection. These nanomaterials have the advantages of high sensitivity, real-time sensing ability, and almost infinite contrast against background. This review focuses on appraising nanotechnologies with the ROS “turn-on” detection mechanism coupled with the ability for broad biological applications. In this review, we highlighted the weaknesses and advantages in prior sensor studies and raised some guidelines for the development of future nanoprobes. It mainly includes singlet oxygen (e (H2O2) . ROS hase (H2O2) , which ie (H2O2) . Therefoin vivo and live cell imaging  and good in vivo utility of this nanoparticle may support its eventual application in clinics (limit of detection (LOD) = 0.5 μM). Higher selectivity has been achieved for the nanoparticle for the detection of H2O2 over other ROS and biologically relevant species (2O2 (LOD = 3.33 μM). Moreover, they fabricated a H2O2-triggered drug release system for heart failure therapy. This system exhibits the potential for different variants of heart failure models to target theranostic treatment  MOF for “turn-on” sensing of H2O2 in live cells (LOD = 0.015 μM). However, it also has moderate response to some other ROS and biologically relevant species, indicating that the selectivity of this MOF material toward H2O2 is a major defect  mechanism was fabricated by Zhang’s group. In this nanoprobe, diphenylphosphine moiety is covalently attached to the surface of the carbon dot; they serve as the PET donor and acceptor, respectively. Subsequently, H2O2 can selectively oxidize the diphenylphosphine to produce the target oxide and prevent the PET mechanism; then the fluorescence will “turn on” (2O2 with a LOD of 84 nM (2O2 detection (LOD = 5 nM). Nanointegration of near-infrared carbon nanodots and peroxalate (P-CDs) with amphiphilic triblock copolymer as bridge can serve as “turn-on” sensors for the detection and imaging of H2O2 , in which, UCNPs and DNA-AgNPs serve as donors and quenchers, respectively. This design results in luminescence quenching of UCNPs using DNA-AgNPs by luminescence resonance energy transfer (LRET). Upon H2O2 introduction, AgNPs can be converted to Ag+, leading to the inhibition of the LRET process and inducing the recovery of upconversion luminescence -nanosheet-modified UCNPs for rapid detection of H2O2 (LOD = 0.9 μM). The MnO2 nanosheets on the surface of UCNPs serve as the quencher. Fluorescence of UCNPs will be recovered after the addition of H2O2, which can reduce MnO2 to Mn2+ and destroy the structure of the MnO2 quencher (2 probe (LOD = 0.87 μM) and a three-in-one stimulus-responsive nanoplatform , respectively, for H2O2 sensing with relatively improved selectivity or sensitivity , is the most deleterious and reactive species of ROS. The general reactivity of the main ROS in biological systems decreases in the order of •OH > 1O2 > H2O2 > O2•−   of the QDs. Consequently, the produced electron–hole pairs could emit strong luminescence through electron–hole recombination. This nanotechnology is demonstrated to have an application in detecting the endogenous release of •OH in living cells  acting as a functional group on the cytosensor is oxidized to ox TMB, a colored product, and can be monitored through colorimetric analysis. Coupled with a rational design, the nanotechnology has been constructed as a convenient method for the sensitive detection of MCF-7 cells (LOD = 20 cells ml−1) ( > O2•− . •OH canlifetime . Therefo 0.3 μM) . In thisng cells . Alternals ml−1) .1O2 has raised vital interest recently as a result of its significance in both chemical and biological systems. 1O2 is the lowest excited electronic state of molecular oxygen but is recognized to be highly reactive. Studies have demonstrated that 1O2 is highly toxic and destroys key biological molecules including proteins, DNA, and unsaturated lipids. Depending on a triphenylphosphonium derivative, the self-assembled nanomaterial has been fabricated for 1O2 “turn-on” detection (LOD = 33–56 μM)  through photonic luminescence enhancements has been developed  ion so as to induce these nanoprobes to become luminescent  in this system functions as a crucial moiety for the fluorescence “turn-on” sensing process. The presence of 1O2 in this system can result in change in the SOSG fluorescence quantum yield, which permits a stronger energy transfer from the SOSG probe to a proximal silver nanoparticle island film located in the near-electric field of the probe. This induces an increase in the target electric current flow, allowing for the sensing of the 1O2 (3–56 μM) . Howeverand ClO− . A strateveloped . The pSiinescent . Alterna the 1O2 .2•− is a by-product of ATP generation processes of the human body microenvironment, which plays a significant role in regulating biochemistry and organic pathology. Furthermore, exposure to excess O2•− would oxidize organisms, biological membranes, and tissues and cause diseases such as hepatitis, cancer, and diabetes (2•−. Silver nanoparticle (Ag NP)-coated carbon quantum dot (CQD) core–shell-structured nanocomposites (CQD@Ag NCs) have been developed for fluorescent sensing of intracellular O2•− (LOD = 0.3 μM) . The addition of O2•− can convert Fe3+ to Fe2+ and recover the fluorescence of carbon dots  nanoparticles with 4-hydroxy-tetramethylpiperidine at the protein tyrosine residue. The nanoparticles function as a metal-free magnetic resonance imaging (MRI) tool for O2•− monitoring . Enhancement of the fluorescence response of the MOF upon stepwise addition of O2•− has been recorded. The mechanism of the fluorescence “turn-on” procedure is recognized as follows: the structural collapse of the MOF in the presence of O2•− can result in the release of the linker (2-((pyridin-4-ylmethyl)amino)terephthalic acid) with the enhancement of the fluorescence intensity of the system  . In CQD@ecovered . Yue andbon dots  , which can elicit potent characteristic surface-enhanced Raman scattering (SERS) signals at 1,142, 1,386, and 1,432 cm−1 and directly enable the detection of ROS through a hemin-catalyzed Fenton reaction (LOD = 26 pM)  has been realized by the Au-PATP-Hemin nanoprobe. In two typical ROS-elevated mice models of allergic dermatitis and tumors, the Au-PATP-Hemin nanoprobe performed well in monitoring inflammation progression and tumor development in a sensitive and quantitative manner.In some conditions, the evaluation of cellular or system total ROS provides helpful information on cell proliferation, metabolism, and tumor detection. Distinct from the design of nanotechnologies for selective sensing of specific species of ROS, these nanomaterials can detect the combined species of ROS by one platform. Several nanomaterials, including PEGylated bilirubin-coated superparamagnetic iron oxide nanoparticles (PEG-BR@SPIONs) , UCNPs-Mnoflakes , multifunoflakes , para-amnoflakes , cyclotrnoflakes , ROS-resnoflakes , and thenoflakes , are desnoflakes . The “tu= 26 pM) . SimultaIn this short review, nanomaterials with the ability for ROS “turn-on” detection have been deciphered from the aspects of both nanotechnology and chemical reaction mechanisms. In general, “turn-on” nanotechnologies are powerful tools with a low detection limit, real-time sensing ability, and almost infinite contrast against background. Future studies for the design of ROS “turn-on” detection nanomaterials should make efforts to improve selectivity, detection limit, and biocompatibility. Another consideration is the accessibility of these nanomaterials. Reagents for the nanomaterial fabrication are commercially available or can be prepared in simple synthetic steps from commercially available building blocks, which will be greatly in vogue. Continuous efforts are poised to develop more powerful nanotechnologies in this promising field to shed light on critical information of ROS in biological systems.


Staphylococcus aureus (MSSA) ocular infections in Taiwan and compared them between community-associated (CA) and health-care-associated (HA) infections. We collected S. aureus ocular isolates from patients at Chang Gung Memorial Hospital between 2010 and 2017. The infections were classified as CA or HA using epidemiological criteria, and the isolates were molecularly characterized using pulsed-field gel electrophoresis, multilocus sequence typing, and Panton-Valentine leukocidin (PVL) gene detection. Antibiotic susceptibility was evaluated using disk diffusion and an E test. A total of 104 MSSA ocular isolates were identified; 46 (44.2%) were CA-MSSA and 58 (55.8%) were HA-MSSA. Compared with HA-MSSA strains, CA-MSSA strains caused a significantly higher rate of keratitis, but a lower rate of conjunctivitis. We identified 14 pulsotypes. ST 7/pulsotype BA was frequently identified in both CA-MSSA (28.3%) and HA-MSSA (37.9%) cases. PVL genes were identified in seven isolates (6.7%). Both CA-MSSA and HA-MSSA isolates were highly susceptible to vancomycin, teicoplanin, tigecycline, sulfamethoxazole–trimethoprim, and fluoroquinolones. The most common ocular manifestations were keratitis and conjunctivitis for CA-MSSA and HA-MSSA, respectively. The MSSA ocular isolates had diverse molecular characteristics; no specific genotype differentiated CA-MSSA from HA-MSSA. Both strains exhibited similar antibiotic susceptibility.This study analyzed the clinical features and molecular characteristics of methicillin-susceptible  Staphylococcus aureus is a major isolated bacterial pathogen that causes various infections in humans [S. aureus is typically categorized as methicillin resistant or methicillin susceptible based on its susceptibility to methicillin. Methicillin-resistant S. aureus (MRSA), a strain resistant to all β-lactam antibiotics, warrants particular attention because of its potentially limited treatment options and its increasing prevalence [n humans . S. aureevalence . MRSA haevalence . HA- andevalence ,4.S. aureus (MSSA) infections are more prevalent than MRSA infections [Although MRSA has received considerable research attention, methicillin-susceptible fections . Accordifections . In addifections . Howeverfections ,8,9,10, fections . Thus, dS. aureus is the most common cause of bacterial keratitis  and conjunctivitis  [S. aureus isolates were MSSA [S. aureus ocular infections [p = 0.040). In our subsequent research, we focused solely on MRSA ocular infections [fection) ,13. Althere MSSA , studiesfections  and obsefections  and perffections . Becausep = 0.006 and <0.001, respectively). Regarding local risk factors, a significantly higher proportion of the patients with HA-MSSA had a history of ocular surgery, and a significantly higher proportion of the patients with CA-MSSA used contact lenses. Keratitis was the most common ocular disease caused by CA-MSSA, followed by conjunctivitis; however, this order was reversed in the HA-MSSA group . Most of the patients with CA-MSSA received outpatient or emergency department treatment (69.6%), whereas most of the patients with HA-MSSA infection received inpatient treatment (56.9%).During this 8-year study period, MSSA strains were isolated from 104 patients. Of the 104 patients with MSSA, 46 (44.2%) were classified as having CA-MSSA, and 58 (55.8%) were classified as having HA-MSSA. SA group . The ratn = 2), ST59, ST338, ST6426, and ST672, as well as one untypeable isolate ( isolate . p = 0.047)  and fluoroquinolones. The CA-MSSA and HA-MSSA strains exhibited similar antibiotic susceptibility levels, except for susceptibility to erythromycin  .Few studies have explored the clinical and molecular characteristics of MSSA infections, particularly ocular infections. In this study, we investigated the clinical and microbiological characteristics of MSSA ocular infections in Taiwan and compared them between CA-MSSA and HA-MSSA ocular isolates, which have rarely been compared in previous studies. We observed that over half of MSSA ocular infections were HA. Keratitis and conjunctivitis accounted for most of the clinical manifestations of the MSSA ocular infections; significantly more patients with CA infections presented with keratitis, whereas those with HA infections more often presented with conjunctivitis. The molecular characteristics of the MSSA isolates were relatively diverse, and no specific genotype differentiated CA-MSSA from HA-MSSA. The antibiograms of the CA-MSSA and HA-MSSA isolates were similar.S. aureus ocular infections [In this study, a higher rate of HA-MSSA than of CA-MSSA was observed in the MSSA ocular isolates (55.8%), which is consistent with the findings of our previous 10-year study on fections  but contfections . As expeS. aureus is the leading cause of bacterial keratitis and is the most common pathogen isolated from patients with conjunctivitis [ctivitis ,13. Maractivitis . Our prectivitis  and the ctivitis .p = 0.064) among pediatric patients, other research on adult patients [S. aureus ocular isolates in the US, and both studies have demonstrated the polyclonality of MSSA ocular isolates. However, Peterson et al. reported that two major USA clonal complexes, CC5 and CC8, were the most frequently detected clones among MSSA and MRSA keratitis isolates. This discrepancy in the molecular characteristics of MSSA ocular isolates indicates geographic variation.Regarding molecular characteristics, the MSSA isolates in this study were polyclonal, and none of the clones exhibited significant differences between the CA and HA isolates. Our study revealed that ST7/pulsotype BA, ST15/pulsotype F, and ST188/pulsotype AX accounted for half of the isolates, which is consistent with the findings of previous studies on MSSA nonocular infections in Taiwan ,19. Howepatients  did not patients  and Petepatients  have invPVL, a bicomponent pore-forming toxin targeting phagocytic leukocytes, is regarded as a marker of CA-MRSA. In this study, we identified PVL genes in seven MSSA isolates 6.7%). The low prevalence of PVL-positive MSSA strains is comparable to that observed in a global clinical trial , which r%. The loIn the present study, the HA-MSSA and CA-MSSA isolates both exhibited high susceptibility to most of the tested antibiotics except for clindamycin and erythromycin. The antibiograms of the MSSA ocular isolates were determined to parallel those of nonocular MSSA isolates in Taiwan. We tested the isolates’ antibiotic susceptibilities to fluoroquinolones, the first-line treatment of ocular infections, and observed that the susceptibility rates to fluoroquinolones all exceeded 90%. This rate is higher than that (approximately 80%) reported by Asbell et al. in their study on MSSA infection in the ocular Tracking Resistance in U.S. Today (ocular TRUST) program . Taiwan’S. aureus, S. epidermidis, and Pseudomonas aeruginosa in vitro [S. aureus, S. epidermidis, and Candida species [The emergence of drug resistance necessitates innovative therapeutic approaches. Recently, new ophthalmic solutions have been developed. Povidone–iodine 0.6% was determined to exhibit higher bactericidal activity than does povidone–iodine 5% ; a commein vitro . Another species . In addi species  reportedIn contrast to the differentiation of MRSA isolates, differentiating between CA-MSSA and HA-MSSA ocular isolates is difficult. In our previous study on MRSA ocular infections, we found that patients with CA-MRSA ocular infections were, on average, younger and more frequently diagnosed with eyelid disorders than those with HA-MRSA infections . The domS. aureus is a common colonizer of the ocular surface. However, all the isolates included in this study were clinical samples collected from patients with active ocular infections. Finally, this study was conducted in a single tertiary-care hospital; therefore, the results might not be generalizable to other populations. However, because our hospital is the largest referral hospital in Taiwan, the results of this study may still generally reflect the epidemiology of MSSA infection. Additional prospective studies involving patients from more hospitals are warranted.Our study has several limitations. First, although the isolates were prospectively collected, the clinical data were retrospectively reviewed. Therefore, the patients might have been misclassified due to incomplete evaluation of risk factors. Nevertheless, we were still able to observe some significant differences between the patients infected with the CA and HA-MSSA isolates. Second, the sample size might not have been sufficiently large, but the correlation between pulsotypes and STs is similar to that recorded in the study on pediatric MSSA infection in Taiwan . Third, The study adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board (IRB) of Chang Gung Memorial Hospital (CGMH), a tertiary medical center in Taoyuan, Taiwan (IRB 107-2346C). The requirement for written informed consent was waived due to the anonymous analysis of the data.S. aureus isolates were prospectively collected from patients with ocular infections and stored in the microbiology laboratory of CGMH. The medical records of the patients were retrospectively reviewed for demographic and clinical information. If more than one isolate was collected from a single patient, only the first was included for analysis. The MSSA infections were classified as HA or CA based on the definitions employed by the CDC since 2000 [From 1 January 2010 to 31 December 2017, clinical nce 2000 . The cliThe patients’ underlying conditions and ocular histories were recorded. The underlying conditions that we screened for were diabetes mellitus, hypertension, pulmonary disease, renal disease, liver disease, malignancy, immunodeficiency, current infection, recent antibiotic use, and alcoholism; we also screened for recent antibiotic use. The ocular histories included contact lens use, ocular surface disease, surgery, and trauma.Based on the ocular structures involved, the manifestations were categorized into seven diagnoses: lid disorder, conjunctivitis, keratitis, endophthalmitis, wound infection, lacrimal system disorder, and others . If a patient was diagnosed with more than one ocular infection, the primary pathology or the most severe diagnosis was chosen.Molecular analysis methods employed in this study included PFGE by SmaI digestion, PVL gene detection, and MLST, with approaches consistent with those described in detail by Lina et al.  and EnriWe evaluated the antimicrobial susceptibility of the MSSA isolates to antibiotics  through the disk diffusion method according to the Clinical and Laboratory Standards Institute’s standards for antimicrobial susceptibility testing . An E tep value of less than 0.05 was considered statistically significant.All statistical analyses were performed using SPSS . The nominal variables were analyzed through a chi-square test, and the continuous variables were analyzed using Student’s t test. The variables are presented either as a mean ± standard deviation or as a percentage. The correlation between clinical presentations and the classification of MSSA infections as HA or CA was measured using Fisher’s exact test. A two-tailed More than half of the MSSA ocular infections included in this study were classified as HA-MSSA. CA-MSSA caused a higher rate of keratitis than did HA-MSSA, whereas HA-MSSA caused a higher rate of conjunctivitis. Although the molecular characteristics of the MSSA isolates indicated that the isolates were genetically diverse, ST7, ST15, and ST188 were frequently observed in the MSSA ocular infections. Both CA- and HA-MSSA strains exhibited high susceptibility to fluoroquinolones in Taiwan. Physicians should be familiar with the epidemiology, spectrum of diseases, and antibiotic susceptibility patterns of MSSA ocular infections in their local areas. Although we could not clearly differentiate HA-MSSA from CA-MSSA in this study, the results provide information that may be used to enhance local public health policy as well as knowledge on epidemic MSSA clones worldwide. Further research should include larger sample sizes and involve more hospitals to deepen the understanding of the molecular characteristics and clinical features of MSSA ocular infections.


Mean serum lysozyme levels was 39.92 ± 55.5 mg/L in the ocular sarcoidosis group versus 10.5 ± 5.8 mg/L (p ≤ 0.0013) in the control group (n = 30). Both tests were elevated in 8/37 (21.6%) patients, elevated ACE and normal lysozyme was noted in 2/37 (5.4%) patients, whereas the proportion of normal ACE/elevated lysozyme was much higher, 23/37 (62.2%). In 4/37 (10.8%) patients, both tests were normal. The mean score of polyclonal activation (N of elevated CF serologies divided by number of viruses to which a patient was exposed) was 0.6 ± 0.33 in the ocular sarcoidosis group versus 0.15 ± 0.2 for the control group (n = 42) (p ≤ 0.00001). Sensitivity and specificity of ACE and lysozyme were, respectively, 27%/96.6% and 83.7%/90%. Sensitivity and specificity of polyclonal antibody activation amounted to 70%/90.4% Conclusion: Lysozyme was found to be much more useful than ACE as a laboratory test to support the diagnosis of ocular sarcoidosis. As shown in a previous study, polyclonal antibody activation appears to be another useful laboratory test supportive of the diagnosis of ocular sarcoidosis.Background: The diagnosis of ocular sarcoidosis (OS) is difficult to establish in the absence of manifest systemic involvement. To help clinicians reach a diagnosis, we convened a group of experts in 2006 ) to set-up clinical criteria for the diagnosis of ocular sarcoidosis. In addition, laboratory investigational tests represent a much-needed adjunct to ascertain the diagnosis. However, many of these tests have low sensitivity and specificity. Purpose: The aim of our study was to evaluate the usefulness of serum ACE, serum lysozyme and polyclonal antibody activation in the diagnosis of ocular sarcoidosis and compare the frequency of increased serum levels of lysozyme and ACE in proven ocular sarcoidosis or in suspected ocular sarcoidosis. Methods: Serum ACE and lysozyme were assessed in these two groups and their means compared to a group of non-granulomatous  uveitis patients. The proportion of elevated serum ACE versus lysozyme was compared in the sarcoidosis patients. Polyclonal antibody activation was measured by establishing exposition of patients to four human commensal herpesviruses  using ELISA or immunofluorescence and in parallel by performing quantitative complement fixation (CF) serologies. The ratio of elevated CF to positive ELISA/immunofluorescence serologies was calculated. The mean of ratios  was compared between ocular sarcoidosis and control groups. Results: Thirty-seven patients (F24/M13) were included in our study including 17 patients with IWOS Level 1 and 2 criteria qualifying for Group 1 (proven sarcoidosis) and 20 ocular sarcoidosis suspect patients. Mean age was 54.52 ± 23.74. Mean serum levels of ACE was 49.17± 29 IU/L in the ocular sarcoidosis group versus 27.4 ± 15.34 IU/L ( The diagnosis of ocular sarcoidosis is difficult to establish. It was the aim of the organization of the International Workshops on Ocular Sarcoidosis (IWOS) to set diagnostic criteria in order to facilitate the diagnosis of ocular sarcoidosis. The first IWOS was held in Tokyo in 2006 and resulted in a set of clinical criteria for the diagnosis of ocular sarcoidosis . DespiteWe included patients with ocular sarcoidosis according to the modified IWOS criteria in a retrospective fashion. Patients were divided into two categories: (1) proven ocular sarcoidosis patients; and (2) ocular sarcoidosis suspect patients. The first category of patients corresponded to Levels 1 and 2 of the IWOS criteria, i.e., biopsy proven sarcoidosis patients with a compatible uveitis (IWOS Level 1) or patients with a compatible uveitis, where the chest X-ray or CT scan revealed the presence of bilateral hilar lymphadenopathy (BHL) and/or parenchymal peri-lymphatic micronodular pattern (IWOS Level 2). mutton-fat KPs ; (2) iris nodules at the pupillary margin (Koeppe) or in the iris stroma (Busacca) ; (3) tra; (7) opt. Items 1The levels of serum ACE and lysozyme were evaluated.Serum ACE activity was measured using FAPGG (N-[3-(2-furyl) acryloyl]-L-phenylalanyl-glycylglycine; Sigma-Aldrich) with a Buhlmann kit . Normal Serum lysozyme activity was measured by using radial immunodiffusion  , with NAThe means of serum ACE and lysozyme values in the ocular sarcoidosis groups (proven and suspected) were compared to a control group of non-granulomatous uveitis (pars-planitis and HLA B27 related uveitis) for which these tests had been performed as part of the work-up before the final diagnosis was known. The control groups were chosen based on the availability of each test.Furthermore, patients were classified into four groups regarding elevation of both tests to compare their proportion: (1) both tests normal; (2) ACE elevated, lysozyme normal; (3) ACE normal, lysozyme elevated; and (4) both tests elevated.Compensatory increase of immunoglobulins as a result of decrease of T cell activity in sarcoidosis has been published in many reports ,4,5,6,7.n = 30 mean age was 41 ± 11.3, n = 47 mean age was 45.2 ± 17.6 and n = 42 mean age was 45.4 ± 17).Among the 1130 patients seen from 2005 to 2020, 168 patients (14.8%) were diagnosed as possible ocular sarcoidosis patients. Applying the inclusion criteria defined in the previous section, 37 patients  could be included . Mean age was 54.52 ± 23.74. The control group mean age was variable  in the control group (n = 30). Mean serum lysozyme levels were 39.92 ± 55.5 mg/L in the ocular sarcoidosis group versus 10.05 ± 5.88 mg/L (p ≤ 0.0013) in the control group (n = 47) (Mean serum levels of ACE were 49.17± 29 IU/L in the ocular sarcoidosis group versus 27.4 ± 15.34 IU/L ((n = 47) .n = 17) presented an ACE level of 46.69 ± 23 IU/L and a lysozyme level of 43± 67 mg/L. The group with suspected sarcoidosis (n = 20) presented an ACE level of 56.93 ± 32.21 IU/L (p = 0.2603 student’s t test comparing to group of proven sarcoidosis) and a lysozyme level of 37.69 ± 47.87 mg/L .The group with proven sarcoidosis (The proportion of ACE and lysozyme of patients having both tests elevated was 9/37 (24.3%) . The pron = 42) (p ≤ 0.00001), showing a large statistically significant difference of increase of polyclonal activation in the ocular sarcoidosis group.The mean score of polyclonal activation was 0.6 ± 0.33 in the ocular sarcoidosis group versus 0.15 ± 0.2 for the control group (n = 17) presented a polyclonal activation ratio of 0.508 ± 0.356. The group with suspected sarcoidosis (n = 20) presented a polyclonal activation at 0.674 ± 0.28  was 86.6% and Negative Predictive Value (NPV) was 77.5% .Ocular sarcoidosis has a heterogenous presentation rendering diagnosis sometimes difficult. A combination of clinical signs, investigational and laboratory tests with the highest possible sensitivities and specificities has to be sought to achieve the highest probability of correct diagnosis. At present, paraclinical investigations are far from being satisfactory. Therefore, a combination of multiple tests is performed to increase the accuracy of diagnosis. Recent studies have been published proposing new potentially more specific and sensitive biomarkers for sarcoidosis such as soluble interleukin-2 receptor (sIL-2R)  or the eOn the contrary, high serum lysozyme was newly included as a criterion separate from ACE for the diagnosis of ocular sarcoidosis in the revised IWOS diagnostic criteria in 2017  and BaarAnother limitation of the usefulness of ACE serum levels as diagnostic test for OS is the abolition of ACE serum activity in patients taking angiotensin-converting enzyme inhibitors (ACEIs) to treat systemic hypertension.  ACE leve.Finally, additional investigational tests are certainly welcome. In a previous study, we found that the role of polyclonal antibody activation of four human commensal herpesviruses, as a result of decrease of T cell activity captured in sarcoidosis granulomas, appeared as a useful marker for the diagnosis of ocular sarcoidosis . Indeed,Ocular sarcoidosis should be considered in the differential diagnosis of any ocular inflammation, especially if granulomatous signs are present, and a laboratory test can be helpful to obtain a more certain diagnosis. Lysozyme seems to be more helpful than ACE in the diagnosis of ocular sarcoidosis. Polyclonal antibody activation to herpes viruses is another useful biomarker.


As communication technology is entering the 6G era, a great demand for high-performance devices operating in the terahertz (THz) band has emerged. As an important part of 6G technology, indoor communication requires multi-beam steering and tracking to serve multi-users. In this paper, we have designed a graphene metasurface that can realize multi-beam steering for directional radiations. The designed metasurface consists of graphene ribbons, dielectric spacer, and metal substrate. By designing the graphene ribbons and controlling the applied voltage on them, we have obtained single-, double-, and triple-beam steering. In addition, we have also numerically calculated the far-field distributions of the steered multi-beam with a diffraction distance of 2 m. Our design has potential applications in future indoor directional 6G communications. Future 6G wireless communication systems have already attracted tremendous attention ,2. TerahDirectional radiation is a key component in terahertz communication systems. There have been several different techniques previously employed to develop multi-beam patterns in different spectra, involving the use of phased array antennas ,8, gradeIn this paper, we designed a tunable graphene metasurface for far-field multi-beam steering and directional beam propagation in terahertz communication technology. It was shown that the multi-beam steering could be engineered by choosing graphene ribbons with different widths and controlling applied voltage on them. As proofs of concept, we designed three devices for far-field beam steering: 1. steering three-beams at angles of −42°, 13°, and 42°; 2. steering two-beams at angles of −42°, 42°and −42°, 13°; 3. steering a single-beam to an angle of 42°. By studying the near-field distributions and far-field radiation patterns of the multi-beam steering, we demonstrated that our metasurfaces have excellent performances of anomalous multi-beam steering. Our work has paved the way for the application of directional indoor communication.2 layer acts as a dielectric spacer. A metal substrate works as a backside reflector. w, p, and s denote the width of graphene ribbons, the structure period, and thickness of the SiO2 layer, respectively. The metal substrate is assumed to be thick enough. Via choosing appropriate structural parameters and applied gate voltage, the designed metasurface may sufficiently interact with incident light and allows us artificially steer the wavefront of the THz beam.There is a possible application scenario presented here for indoor THz communication. As shown in σintra and σinter are related to the electron-photon scattering process within the band and the interband transition, respectively. At room temperature T = 300 K, the interband transition can be ignored due to the Pauli exclusion principle, and the conductivity of graphene can be simplified to a Drude-like model [e is the electron charge, EF is the Fermi energy level, ћ is the Planck constant, τ is the relaxation time, which is 10−12 s in this work [t is the thickness of the graphene which is 0.35 nm in this work, ε0 is the permittivity of free space. From the above equations, it is clear that the Fermi energy level of graphene can be easily modulated by the gate voltage [fv = 106 m/s, and V is the applied electric potential. So once we know the Fermi energy level of graphene, we can determine the corresponding voltage according to the formula. Due to the tunability of permittivity of graphene, we can achieve dynamic modulation of the beam deflection by designing a metasurface with a specific phase profile.To investigate the designed structure, numerical simulations have been performed. In our model, the graphene ribbons are treated as 2D conductors. Its optical properties can be described by the conductivity uctivity :(1)σg=σike model :(2)σ(w)=his work ,42,43. Whis work :(3)ε(w)= voltage . In genew) and the thickness of the dielectric layer (s) change, respectively. In these cases, the Fermi energy level of graphene is 0.64 eV. The refractive index of SiO2 is 1.4 [p) is 3 μm. The drude model is applied to describe the optical property of a gold-backed reflector with a thickness of 2 μm. The light source is a normal incidence with TM polarization. When the width of the graphene ribbon is near 1 μm, the reflection amplitude varies significantly and phase change almost covers 0–2π, showing the characteristic of plasmon resonance. It can be also observed that the reflectivity and reflection phase will change periodically with the dielectric layer thickness, which may be attributed to Fabry−Perot resonance in the dielectric layer. Herein, we can treat the designed structure as an asymmetric Fabry−Perot resonator, where the graphene ribbons and metal substrate act as front and back reflectors, respectively. According to these results, we can choose a dielectric layer with specific thickness regarding the required reflectivity and reflection phase.As a proof of concept, we designed a metasurface to operate at 12.32 THz. The operating frequency of the proposed graphene metasurface can be easily reduced by scaling the size of the graphene metasurface as graphene strongly interacts with an electromagnetic wave with low loss in the terahertz regime. We used the finite element method to simulate the performances of the unit cell and the designed metasurfaces. 2 is 1.4 , and the2 layer thickness of 3 μm. w) and Fermi energy level of the graphene ribbon, respectively. Due to the Fabry–Perot resonance between graphene ribbons and metal substrate, the continuous 2π phase modulation of reflection can be achieved, further demonstrating the desired metasurface can be constructed by simply choosing the values of w and FE.In this work, we chose the SiOni denotes the refractive index of the medium in the incident region, iθ is the angle of incidence, rθ is the angle of reflection. When the incident light is normally incident and the refractive index of the medium in the incident region in = 1, the reflection angle can be written as:Based on the results above, we can achieve deflection of the reflected beam at the frequency of 12.32 THz. The designing of the metasurface is mainly based on the generalized Snell’s law, in which phase gradient  formula :(4)sin(θAccording to Equation (5), the reflection angle, i.e., the beam steering direction is related to the phase gradient when the incident wavelength is determined. We may achieve multi-beam steering for directional radiations as if we introduced different phase gradients to the metasurfaces. Using this concept, we designed a metasurface to control the normal incident waves and generate multi-beam reflections. We chose 102 graphene ribbons with a period of 3 μm. For the normal incident TM polarized light frequency of 12.32 THz, we first implemented three kinds of phase gradients of −π/6, π/18, and π/6 with 26, 37, and 39 unit cells, respectively. Therefore, the theoretical deflection angles are −42°, 13°, and 42°, which can be calculated from Equation (5). The unit cells are selected appropriately to achieve corresponding phase shifts according to Ex, we can easily see the anomalous reflection of the two asymmetric beams at the directions with angles of about −42° and 13°. Similarly, by selecting 102 graphene ribbons with a phase gradient of π/6, we achieved a single beam deflection with an angle of about 42°, as shown in FE. From When we fix the structure parameters in In the following, we calculated the far-field radiation patterns of our designed metasurfaces. Bandwidth is a key feature to evaluate the performance of communication devices. To demonstrate, we have also simulated beam deflection in the frequency range 12–12.5 THz, and here we give the far-field radiation map for the single-beam case, as shown in E is the electric field,  are the coordinates on the metasurface plane, k0 is the wave vector of the incident light in the vacuum, λ is the incident wavelength.To demonstrate the performances of our designed metasurface, we applied Fraunhofer’s diffraction formula to simulate the amplitude distribution of multi-beam along the x-direction at a propagation distance of 2 m. The Fraunhofer formula is written as:In In summary, we introduced a dynamically tunable metasurface based on graphene ribbons for multi-user indoor communication. By properly choosing the width of the graphene ribbons, the continuous 0–2π phase coverage can be achieved. Similarly, the desired phase shift can be obtained by adjusting Fermi energy levels applied to the graphene ribbons. Simulation shows that the designed metasurface can achieve dynamic adjustable multi-beam far-field anomalous reflection. From the near field distributions and the far-field radiation patterns, we can clearly observe the deflection of three beams, two beams, and a single beam. We also numerically calculated the field distribution of multi-beams at a distance of 2 m away, and the results demonstrated that the beams are separated into beams with different propagation angles. Overall, we have designed a metasurface that provides a new method to achieve indoor directional communication.


Atomic force microscopy (AFM) and scanning electron microscopy (SEM) revealed that these defects are responsible for the inhomogeneous aggregation of antibodies and influenza viruses over the functionalized graphene surface. Non-uniform aggregation is responsible for a weak non-linear logarithmic dependence of the biosensor response versus the virus concentration in PBS. This feature of graphene nano-arrangement affects the reliability of detection of extremely low virus concentrations at the early stages of disease.In this study, we discuss the mechanisms behind changes in the conductivity, low-frequency noise, and surface morphology of biosensor chips based on graphene films on SiC substrates during the main stages of the creation of biosensors for detecting influenza viruses. The formation of phenylamine groups and a change in graphene nano-arrangement during functionalization causes an increase in defectiveness and conductivity. Functionalization leads to the formation of large hexagonal honeycomb-like defects up to 500 nm, the concentration of which is affected by the number of bilayer or multilayer inclusions in graphene. The chips fabricated allowed us to detect the influenza viruses in a concentration range of 10 Two-dimensional (2D) materials are characterized by their strong interplanar bonding but weak interplanar interaction. Interfaces between neighboring 2D layers or between 2D overlayers and substrate surfaces provide intriguing confined spaces for chemical processes, which have stimulated a new area of “chemistry under 2D cover”. The interaction between electrons and the honeycomb carbon lattice causes the electrons to behave as massless fermions, which give rise to novel physical phenomena such as anomalous room temperature quantum hall effect, extraordinarily high carrier mobility, high surface area per unit volume, and a low-noise [The rapid spread the coronavirus disease 2019 (COVID-19) during the pandemic and regularly occurring epidemics of influenza, which have killed hundreds of millions of people and caused significant damage to the global economy, have shown the need to create highly sensitive biosensors that allow quick (within minutes) detection of extremely low concentrations of antigens (viruses) at early stages in these diseases. The fabrication of such biosensors would make it possible to find out the mechanism of the spread of COVID-19. Recent reports have proposed graphene as a prospective material for these biosensors ,4,5,6 thow-noise ,8,9,10. When it comes to the registration of influenza viruses and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, several reports have shown the great promise of the application of chips based on graphene films with two contacts, i.e., graphene resistances [Significant improvements in the detection of low concentration of influenza viruses and COVID-19 by graphene biosensors have been achieved recently. Some works ,7 reportReproducibility issues relating to the structural and physicochemical properties of graphene resistances are discussed in ,7,11,12.The commonly accepted concept of viral biosensor production is based on the creation of conditions for the antibody–antigen (virus) immune reaction on a graphene surface. It can include controlled treatment  of the graphene surface to create covalent bonds that ensure the occurrence of selective chemical reactions of the attachment of biomolecules (antibodies) ,7,8,9,10Numerous techniques such as vapor deposition, epitaxial growth, mechanical and chemical exfoliation, and the thermal destruction of the silicon carbide surface have been explored for achieving desired properties in graphene . A shortIn this paper, we used graphene films obtained by thermal destruction of the SiC substrates surface for biosensor chips used to detect the influenza viruses. We show the changes in the resistance, low-frequency noise amplitude, and graphene nano-arrangement reflected in surface morphology during the main stages of biosensor chip development . The uniformity of the distribution of these changes over the chip area was also investigated by probe methods of analysis, as well as by a low-frequency noise technique.The main stages of graphene-based biosensors are presented in In our experiments we used 4H-SiC substrates with a minimum misorientation angle (α ~ 0), and the growth was carried out on the (0001) ± 0.25° orientation (Si face). We used semi-insulating substrates. For the successful development of graphene growth technology, a necessary condition is the high-quality preparation of the SiC surface, which reduces the effect of contamination and surface inhomogeneity on the sublimation process. Pre-growth etching in a hydrogen atmosphere was used for preliminary cleaning of the SiC substrate surface. The essence of the technology lies in the high-temperature heating of the SiC substrate in a hydrogen atmosphere. At high temperatures, free carbon formed on the SiC surface binds with hydrogen to form volatile chemical compounds. We used a gas mixture containing argon (volume fraction 95%) and hydrogen (volume fraction 5%). Then, the growth of graphene on the SiC surface was performed at a temperature of 1700–1800 °C in an argon atmosphere (720–750 torr). The growth process was carried out in a graphite crucible with induction heating.2.After the graphene films growth, a conventional photolithography process was used to pattern the graphene/SiC chips. The chips were processed from several samples of graphene films formed by thermal decomposition of semi-insulating 4H-SiC. Details on graphene film processing and the mounting of chips on holders can be found elsewhere . This stTo provide sensing ability, graphene functionalization is usually accomplished by using various covalent and noncovalent approaches ,8. Graph6H5NO2) and (2), the subsequent reduction of the nitrophenyl groups to phenylamine groups  by the method of cyclic voltammetry (CV). All CV experiments were performed in a conventional three-electrode cell with an Ag/Ag+ (or Ag/AgCl) reference electrode, a platinum wire counter electrode, and a graphene/SiC chip as the working electrode. The three-electrode cell had a hermetic lid allowing the electrolyte and the space above it to be purged by dry Ar to remove traces of the moisture from the cell and the electrolyte.In this work, the process of the functionalization of the graphene surface in chips was carried out in two stages: (1) the formation of covalent bonds during the deposition of nitrophenyl groups .At the first CV stage, the nitrophenyl groups were attached to the graphene surface. For this, a graphene chip assembled on a holder was immersed for 1–2 min in a non-aqueous electrolyte based on a mixture of 2 μM 4-nitrobenzenediazonium tetrafluoroborate (4NDT) and 0.1 M tetrabutylammonium tetrafluoroborate (TBATF) in acetonitrile  solution in order to reduce the nitrophenyl groups to the phenylamine groups on the graphene die surface. Details on the graphene functionalization process can be found in .−16 g/mL to 10−9 g/mL.After surface functionalization, all chips were incubated in a solution containing influenza A (or B) antibodies for 3 h at 37 °C, followed by a single wash in PBS. The detection of influenza antigens (viruses) in PBS was then carried out. Concentrations of influenza virus in PBS solutions ranged from 10For antibody immobilization on the functionalized graphene surface, we used the same concentration of antibodies diluted in the buffer solution in all experiments. Anti-NP monoclonal antibodies were dissolved at 200 μg/mL in PBS. The concentration of the antibodies was higher than the possible places with covalent bonds suitable for the antibody’s attachment. We did not observe changes in conductivity after the antibody conjugation.The following strains of influenza viruses used in the experiments were obtained from the collection at the museum of viruses of the Smorodintsev Research Institute of Influenza, Russia: influenza virus A/California/ 07/09 (H1N1pdm09) and influenza virus B/Brisbane/46/15. All experiments with viruses were carried out in a BSL-2 facility at the Institute of Influenza by its employees, who are co-authors of the work. All permits were in place.Lysates of purified virus concentrates were used as an analyte. The lysates were pre-pared by diluting viruses in a lysis buffer , followed by a freeze-thaw step. Such viral lysates mainly contain destroyed virions. Therefore, the concentration of viruses in the lysates was assessed by measuring the total viral protein with the modified Lowry method, using the RC DC Protein Assay Kit . Analyte solutions were prepared by tenfold dilution in PBS. The analytes were incubated at room temperature.The biosensor concept in our studies is based on antigen–antibody immunoreaction on the graphene surface. The selectivity or specificity for sensing performance is mainly due to nature of the immunoreaction: only related (matched) antigens and antibodies participate in the interaction and can change the state of the graphene. However, other viruses can influence the biosensor response via mechanisms other than the immunoreaction.In this study, we did not use a special passivation of the graphene surface. The response of biosensors with immobilized antibodies was investigated under the conditions of diluted solutions of related antigens in order to determine the influence of the graphene surface on the detection process.During the detection process, a direct current voltage (20–80 mV) was applied to the graphene chip coated (immersed) with the influenza antibody, and the chip was immersed in a PBS diluted solution of influenza virus (antigen) for 30 s. The influenza antigen chemically attaches to the influenza antibody which results in a change in the resistance of the graphene channel, which can be promptly detected by the passing of a current through the graphene/SiC chip. Thereafter, the chips were pulled out, washed in pure PBS solution, dried, and immersed again in another PBS solution with a different influenza virus concentration.Current–voltage (I–U) characteristics and low-frequency noise spectra containing information on the defective system state, which depict the quality of the material, were studied in the chips after each stage of the biosensor fabrication. The surface morphology and the surface potential distribution were monitored by atomic force microscopy (AFM) and Kelvin probe force microscopy (KPFM). In addition, scanning electron microscopy (SEM) was used to visualize the attachment of antibodies and influenza viruses to the graphene surface.www.tipsnano.com, accessed on 22 December 2021). A resonant mode of operation was used in the work. The AFM probe knocks on the surface scanning frequency 0.6 Hz. The scanning speed was approximately 1.3 μm/s. The stiffness coefficient of such a cantilever is 3.5 N/m, the radius of curvature is less than 10 nm, and the scanning field size is 256 × 256 points.AFM and KPFM measurements were carried out on an Ntegra AURA setup . AFM studies were carried out using the HA_FM cantilever  and subsequently measured by an SR 770 FET NETWORK Analyzer . The background noise of the preamplifier did not exceed 4 nV/√Hz at 1 kHz, which is approximately equivalent to the Johnson–Nyquist noise of a 1000-Ω resistance. SEM analysis of the chip surface was carried out by a JSM 7001F microscope  in the secondary electron mode at an accelerating voltage of 5 keV and a beam current of 12 pA.The I–U characteristics were measured using the KEITHLEY 6487 power source. The power spectral density of voltage fluctuations was measured for the frequency range of 1 Hz to 50 kHz. The studied samples were connected in series with a low-noise load resistor R, the resistance of which varied from 100 Ω to 13 kΩ, depending on the current passing through the chip. The voltage fluctuations S2 were analyzed with subsequent plotting of Raman maps of spectral lines parameters. A YAG: Nd solid-state laser with a wavelength of 532 nm was used as an excitation source.Raman spectroscopy measurements were carried out at room temperature in the backscattering geometry using a T64000 spectrometer  equipped with a confocal microscope. The laser power of a YAG:Nd laser with a wavelength of 532 nm was limited to 1.0 mW in a spot 1 μm in diameter to prevent the damaging and modification of the graphene films. Along with local measurements, sample areas of 10 × 10 μmU before and after functionalization. The chips from several plates were investigated. The first five characters in the chip notation in The I–U characteristics of all chips under study remained linear at each stage of the biosensor development. −1 were dominated by sharp G and 2D lines characteristic of monolayer graphene [−1 corresponding to the buffer layer [−1 appeared in the spectra. This is attributed to the appearance of defects in the graphene’s crystal lattice. We did not observe any significant shift or broadening of the G and 2D lines after the functionalization of the chips.The presence of graphene films on the SiC surface was confirmed by Raman spectroscopy, as shown in graphene  and wideer layer . After f−1, this line has an asymmetric contour corresponding to the envelope of four Lorentzians, which is a fingerprint of bilayer graphene [Analysis of the full width at half-maximum of the 2D line (FWHM2D) distribution before and after functionalization allowed us to analyze the distribution of mono- and bilayer graphene areas on the surface of the chips. graphene . One canU ~ 1/f was observed before and after functionalization. This type of dependence is typical in graphene. The dependence indicates that the noise is determined not by uniformly distributed single defects in the material but by a system of defects [The low-frequency noise spectra of graphene chips are shown in  defects ,25. A hi defects .U grew. Meanwhile, the chips from Group 2 showed no significant changes in these parameters. The changes in Raman spectra for the chips in Group 1 are similar to those presented in [The resistance of chips in Group 2 is noticeably lower than that in Group 1 before functionalization . An oppoented in , while t2, 8 × 8 μm2, and 2000 × 2000 nm2. The most informative in terms of nano-arrangement are the 2000 × 2000 nm2 fields. It should be noted that the 2000 × 2000 nm2 fields are the most informative in terms of studying nano-arrangement because the chips in Group 1 typically had the smallest bilayer inclusions. We employed AFM to clarify the behavior of the bilayer graphene inclusions. Studying the surface morphology of the graphene chips of these two groups revealed significant differences in the nature of their nano-arrangement both before and after functionalization, as illustrated in 2. The Group 2 chips had a rather higher AFM profile, up to 10–15 nm, and inclusions occupied a larger area  by cyclic voltammetry leads to decreasing resistance values, since aminophenyl groups have weaker acceptor properties than nitrophenyl groups.The covalent binding of nitrophenyls to graphene films is known to lead to a remarkable decrease in conductivity. This happens because of a reduction in graphene aromaticity due to the transformation in hybridization of carbon atoms from spThus, graphene nano-arrangement, in addition to its functionalization, can contribute to a decrease in graphene resistance. All the obtained results confirm that functionalization is accompanied by an increase in graphene defectiveness due to the formation of large honeycomb-like defects up to 500 nm in plane. At the same time, small inclusions of bilayer graphene disappear or decrease noticeably. This phenomenon might be related to the reduction properties of phenylamine groups. These results allow us to suggest that shallow bilayer inclusions contain nonequilibrium phases of weakly oxidized graphene. SEM images of the surface morphology of a graphene chip after functionalization, immobilization of influenza B antibodies, and antibody–virus B antigen immune reaction are presented in Insert 1 in The further stages of biosensor fabrication  were studied mostly on the chips of Group 1. Significant changes in resistance and low-frequency noise, which are comparable to changes after functionalization, were not observed after these stages. As a result, we chose probe methods to study the chips after each of these stages.−16 g/mL to 10−10 g/mL. 2 close to 1 (0.96) in chips from Group 1 with lower concentration of honeycomb-like defects. A similar dependence was observed when the response of a graphene-based biosensor used to contact with solutions of egg albumin in PBS was studied [The conventionally accepted method described in  studied ,29. It s studied ,12. The We assumed that aggregation and graphene nano-arrangement might influence the trend of the concentration dependence of detected signal. Therefore, we studied AFM profiles of functionalized graphene surfaces after the immobilization of influenza virus antibodies and after antibody–antigen influenza virus immune reactions. Probe methods revealed the aggregation of antibodies and their non-uniform distribution over the graphene surface of the chips ; these wBecause of this aggregation, it was difficult to estimate the lateral dimensions of a single antibody. Its vertical dimension was about 15–20 nm, while in clusters it was more than 20–25 nm, as can be seen in The maximum lateral dimension of the antigen aggregates reached 5 μm and their height reached up to 250–300 nm. An enlarged image of one of the aggregates with a lateral dimension up to 5 μm is shown in inset 2 in The aggregation of antibodies and viruses seems to be one of the reasons for the weak dependence of the current passing through the chip (chip response) on the concentration of viruses in PBS solutions, as shown in 2 AFM scan images in When the viral concentration is high and it is necessary to register the presence of the virus, aggregation becomes a positive factor. Recently, a hexagonal honeycomb-like structure was created artificially by etching or laser-engraving . StudiesThe shape of defects can be hexagonal. At the same time, light ball-like aggregates of antibodies are located either in the dark areas or at the borders of the light regions, i.e., on the nano-step a.Inset 2 in The study has shown that a graphene monolayer with 5% of bilayer inclusions, grown on SiC substrates, could be the basis for creating influenza virus biosensors. A set of diagnostic methods that includes SEM, AFM, Raman spectroscopy, and low-frequency noise measurements allow one to characterize the properties of graphene chips during the main stages of development. We found significant increases in the number of defects as well as a growth in conductivity in graphene chips after functionalization. We associate the observed phenomena with both the attachment of aminophenil groups and changes in nano-arrangement of the graphene. The changes in the graphene nano-arrangement are reflected, in particular, in the features of the surface morphology. Functionalization is accompanied by the formation of large honeycomb-like defects up to 500 nm in plane. At the same time, small inclusions of bilayer graphene disappear or decrease noticeably. An increase in the amount of bilayer or multilayer inclusions in graphene leads to a noticeable growth in the number of honeycomb-like defects. SEM and AFM measurements revealed that these defects facilitate the aggregation of antibodies and influenza viruses. Furthermore, they depict a non-uniform distribution of aggregated antibodies and influenza viruses over functionalized graphene films.−16 g/mL to 10−10 g/mL in PBS.The features of graphene nano-arrangement affect the reliability of detecting extremely low concentrations of viruses during the early stages of diseases. They may also be the cause of observed weak non-linear logarithmic dependence of biosensor response versus the virus’s concentration. A decrease in the concentration of honeycomb-like defects in graphene made it possible to achieve an almost linear-logarithmic dependence of the response of the graphene biosensor versus the virus concentration in the range from 10Thus, the control of graphene nano-arrangement is important to reduce the effect of viral aggregation and to create extremely sensitive biosensors for influenza viruses.


This study provides evidence supporting the operation of a novel cognitive process of a somatic seat of attention, or ego-center, whose somatic location is under voluntary control and that provides access to differential emotional resources. Attention has typically been studied in terms of what it is directed toward, but it can also be associated with a localized representation in the body image that is experienced as the source or seat of attention—an aspect that has previously only been studied by subjective techniques. Published studies of this phenomenon under terms such as egocenter or self-location suggest that the seat of attention can be situated in various ways within the experienced body, resulting in what are here referred to as different attentional stances. These studies also provide evidence that changes in attentional stance are associated with differences in cognitive skill, emotional temperament, self-construal, and social and moral attitudes, as well as with access to certain states of consciousness. In the present study, EEG results from multiple trials of each of 11 specific attentional stances confirmed that patterns of neural activity associated with the voluntarily control of attentional stances can be reliably measured, providing evidence for a differential neural substrate underlying the subjective location of the seat of attention. Additionally, brain activation patterns for the attentional stances showed strong correlations with EEG signatures associated with specific positive emotional states and with arousal, confirming that differential locations of the seat of attention can be objectively associated with different emotion states, as implied in previous literature. The ability to directly manage the seat of attention into various attentional stances holds substantial potential for facilitating access to specific cognitive and emotional resources in a new way. Historically, the sense that there is a beam of attention emanating from oneself to the object of attention (such as a chair) was so strong in the classical era that philosophers understood it as a beam of light emanating from the eyes and reflecting back information about the object of regard. It is explicit that this beam of light/attention can be directed at will toward any object in the field of view, as is the subject of numerous attentional experiments in the modern era. Of course, such concepts are all metaphorical imagery for neural excitation processes in the brain, with a target in the neural representation of the location of objects in space, that is enhanced by a directed neural process under the voluntary control of the location of interest.egocenter or self-location, without reference to its possible importance for the study of attention. This study describes the body region where attention is directed from as the seat of attention, to distinguish it from what attention is directed toward, which is the target of attention. Just as one's perspective on objects in the environment changes while walking through a forest or down a city block, a change in the seat of attention creates a shift in attentional perspective, resulting in a different attentional stance—a variable that has been shown to affect state of consciousness, emotional temperament, self-construal, and social and moral attitudes.This kind of focal attention has typically been studied in terms of what it is directed toward, but the hypothesis for the present study is that this beam is also perceived to have an adjustable source in the body from which it emanates, like a fireman holding their hose over the shoulder or down by the waist. The body location from which attention is sensed to project—typically either the head or upper torso—has been the subject of a small number of studies under terms such as a location from which attention is directed. For this study, “seat of attention” is the preferred term. However, all previous evidence for this psychological construct has been based on subjective report of its existence and perceived location in the intrapersonal and peri-personal body space.The sense of where I am located is associated in a generalized way with my overall body's location in space, but within the body some locations are more strongly associated with the felt sense of the located self , or at the level of the upper torso, with a minority of cases near the neck or lower face  being able to move their seat of attention from one designated location within the body  to another; (b) being able to move their seat of attention to places other than the head and chest; and (c) being able to shift between focused and diffuse attentional stances at each location;Each attentional stance will show a differential scalp activation pattern;Within-participant inter-run correlations on the same attentional stance will be positive, indicating consistency in brain activity associated with each attentional stance;EEG signatures associated with attentional stances will show an increase in higher frequencies as the seat of attention is situated higher along the body's superior-inferior axis or between focused and diffused stances;The signature of some attentional stances will correlate significantly with those of positive emotions and/or affective dimensions;Attentional stances that soften the sense of boundaries or extend beyond the boundaries of the physical body will be associated with positive emotional states;Attentional stances that do not extend beyond the boundaries of the physical body will not be associated with positive emotional states;Only focused attentional stances aligned with the body midline will be strongly correlated with emotion-related arousal;Diffuse and focused stances will display inverted correlational relationships to positive emotions and arousal.Eight participants (5F/3M) with a mean age of 54 (range: 32–71) who reported facility in self-regulation of their attentional stance were measured with a 128-lead EEG array while assuming 11 specific attentional stances. The sample size is modest due to the difficulty of identifying individuals capable of rapidly moving through multiple attentional stances, which is rather more demanding than the common use of self-regulation for simplified access to meditative, embodied, or performance-related states. Experimental procedures were reviewed and approved by the California Institute of Integral Studies Human Research and Review Committee, and participants provided written informed consent.Some stances required the seat of attention to be focused in a particular body location, while in others it was required to be diffused over an extended region of body-relative space. Participants completed three 20 s trials of each attentional stance. The 127-channel EEG was recorded continuously throughout the experimental session (see below). The trial protocol was identical for each participant, except that each set of stances arranged in a different randomized sequence to eliminate order effects. Participants were instructed to assume one attentional stance at a time for 20 s, asked to indicate when each attentional stance had been achieved, marking the time at which the 20 s sample was extracted from the continuously-recorded EEG.Normally, most people in the schooled Western culture think of their attention as emanating from somewhere in the middle of the head, but different social contexts may move it to different locations in the body. For example, when dancing or listening to rock music, the seat of attention may naturally shift to somewhere in the abdomen. Experience with individuals interested in controlling their attentional stance shows that they can report moving the seat of attention to a wide range of bodily locations, and also of controlling whether it is locally focused or more broadly diffused. Inspired by a small number of published reports of self-location extending beyond the physical body , which incorporates 128 electrodes distributed around the head and face, so that the electrode net spans all regions of the scalp overlying any part of the brain . The impEEG results were analyzed by a normalized frequency-band analysis that was validated by a frequency-domain principal components analysis. One approach to frequency-based analysis is to apply a principal components analysis (PCA) to the frequency spectra across conditions to identify the primary components controlling the differential EEG activity during the attentional tasks. For this, the data were first filtered with a filter proportional to frequency (f), because EEG spectra tend to fall with a 1/f spectrum up to about 100 Hz, while the signal-to-noise ratio tends to remain roughly constant over the same range. The PCA was then computed by the Matlab Singular-Value Decomposition (SVD) function. The eight components contributing the most to the PCA variance are plotted in Inter-run correlations of the EEG frequency spectrum amplitudes were computed, for each combination of frequency band and attentional state, across the three runs for each participant. Blink artifacts were avoided by subdividing the runs into 2s epochs and restricting the inter-run correlation analysis to samples in which the epochs were blink-free.The EEG scalp patterns of 10 positive emotions and 4 related affective dimensions were measured by Hu et al.  while waFor the emotional analysis, the patterns of activation over these 32 scalp locations for each of 10 emotions and 4 affective dimensions were correlated with the patterns of activation across these locations in the present study, for each of the 11 attentional stances in each of the 5 EEG frequency band (a 14 × 11 × 5 correlation matrix).p ≤ 0.002, a level that would allow less than one false positive per frequency band.The approach taken to the group-level statistical analysis is to rely on individual significance levels for each correlation of the pattern of scalp activation for each of the 10 emotions with that in each frequency band for each of the 11 attentional stances, with correction for multiple applications. Across 32 scalp locations, this correction provides for correlations ≥0.5 being treated as significant at p < 0.001, or Bonferroni criterion of p < 0.05 corrected for 50 applications of the test). This approach combines statistical rigor with a flexible analysis capability for complex data structures. Given the sample of 8 participants, this criterion corresponds to an effect size of 1.06 for any reported result, which is well beyond Cohen's . Transitions between attentional stances typically required <1 min to achieve as indicated by the participants with a slight nod of the head following the instruction about which state to target at the beginning of each trial, including some seconds allowed for stabilization of the stance. All participants reported the ability to shift to each of the 11 different attentional stances prescribed for the study, including locations in the lower torso (Hypothesis 1b), and were able to enter both focused and diffuse attentional stances (Hypothesis 1c).Each attentional stance showed a differential pattern of scalp activation in at least one frequency band (Hypothesis 2); the average responses across the three repeats for all 127 active electrodes under the 11 attentional stances in each of 9 EEG frequency bands are shown in Multiple analytic approaches found no discernible superior-to-inferior or focused-to-diffused pattern across the EEG frequencies (Hypothesis 3), suggesting that the seat of attention is encoded elsewhere than near the somatosensory cortex. Moreover, within-participant inter-run correlations for each attentional stance were positive for all measured frequency bands. Inter-run correlations were lowest in the alpha bands but higher than 0.5 in all bands above the alpha frequencies (Largely in line with expectations (Hypothesis 6), four of the five attentional stances expected to soften the sense of boundaries through diffusion or extension beyond the bounds of the physical body were strongly correlated (≥0.5) with at least one positive emotion in one band . Only two of six attentional stances focused within the bounds of the physical body were associated with positive emotions, which was partially in line with predictions . As predicted (Hypothesis 8), the three attentional stances associated with arousal were aligned with the body midline .Figure 7; Hypothesis 9); this was also the case in the gamma band for two of these conditions . For example, when attention was focused at the core of the whole body, beta activity correlated positively with four emotions  and negatively with six emotions ; when attention was diffuse throughout the whole body, beta activity correlated in precisely the opposite way—negatively with the four emotions that showed positive correlations with the focused state, and positively with the six emotions that had negative correlations in the focused state.In three of the four conditions where a similar seat of attention was used for both a focused and diffuse attentional stance , the focused and diffused conditions correlated with positive emotions in an inverse way in the beta band . In three of the four locations of attention where both focused and diffuse stances were used, the correlation with emotion-related arousal was inverted. That is, in the beta and gamma bands three focused attentional stances  correlated positively with arousal levels associated with positive emotions, while for three corresponding diffuse attentional stances  correlations with arousal were negative to approximately the same degree.A similar inverse relationship between focused and diffuse attentional stances was observed relative to arousal . This is also the case with the diffuse attentional stances , except that as noted the profiles of diffused attentional stances are inversions of those for the focused attentional stances.Given that there were no such inverse correlations in theta, low alpha, or high alpha bands, and that focused and diffuse attentional stances situated in the chest did not show the predicted inverse correlations, Hypothesis 9 was only partly confirmed.p < 0.0001 for the 31 degrees of freedom of the scalp locations, implying no false positives over the 770 tests conducted for Of the 11 attentional stances, six showed strong positive correlations (≥0.5) in at least one frequency band with the EEG signatures reported by Hu et al.  associatThe specific positive emotions that were found to show a pattern correlation with the attentional stance patterns are tabulated in source of this attentional focus, the felt location of the perceiving self, is always at the same bodily location (such as the center of the head), or whether its felt location can be relocated under voluntary control. This study had two major goals. The first was to provide objective evidence in support of the concept that there is an identifiable seat of attention located somewhere in intrapersonal space that can be voluntarily controlled. The second was to utilize the pattern of EEG activation associated with each attentional seat, or stance, included in the study to identify the emotional tone accompanying each one.Selective attention is well-known to be directable to specific targets in the field of awareness, both exogenously and under voluntary control. What has not been previously established is whether the The results offer the first evidence that the self-regulation of this seat of attention in the felt experience of body-relative space can be used to voluntarily access specific attentional stances, and that these stances can be situated in areas of the body other than the head and chest. These attentional stances were reliably associated with specific patterns of brain activation, and activation patterns differed between stances. Attentional stances therefore appear to correlate with stable patterns of neural activity. This suggests that the construction and regulation of the seat of attention is a novel cognitive process not previously assessed.The fact that regulation of the seat of attention appears to directly affect access to emotional and cognitive resources is in line with previous findings that the subjective experience of self-location served this function  in at least one frequency band. This suggests that selection and use of attentional stances associated with different cognitive and emotional resources may offer a simplified way to access desirable mental and emotional states and their associated resources. Since participants in this study were typically able to relocate attention from one stance to another in less than a minute, self-regulation of the seat of attention may provide a rapid and efficient way to access such resources.Applications of the findings will require additional research to clarify the mechanisms by which self-regulation of the seat of attention impacts cognitive, emotional, and attentional states. However, in order to guide the development of future hypotheses it may be useful to consider ways in which attentional stances may be related to various mental states.For example, typically complex or high-performance states such as mindfulness or flow states are accessed by indirect means and learned slowly. Training in state mindfulness in a mindfulness-based stress reduction context, while effective, will often require 25–30 h of instruction plus hours of individual practice before the target state can be reliably accessed and retained, and the skill maintained. This is likely in part because the mindful state is taught through inductive learning, as a byproduct of repeated exercises in a group context where a leader is modeling the mindful state. By contrast, training based on the attentional stance of the mindfulness state could provide greatly accelerated learning and expedited access to such states.As a specific example, in Marolt-Sender's  study wiSelf-regulation of the seat of attention to head or chest locations may also provide direct access to cognitive and affective states associated with head-located or chest-located attentional stances. Evidence from prior work suggests that the seat of attention, corresponding with self-location, may be a variable in cognitive efficacy, emotional temperament, self-construal, and social and moral attitudes  for correlations between patterns of scalp activation associated with specific attentional stances and those associated with positive emotions in a different study  and significance (up to The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Human Research Review Committee, California Institute of Integral Studies. The patients/participants provided their written informed consent to participate in this study.GH, LL, and CT contributed to the conception and design of the study. LL and CT performed the statistical analysis and wrote the sections of the manuscript. GH wrote the first draft of the manuscript. All authors contributed to the manuscript revision, read and approved the submitted version.This work was funded from NSF/SL-CN 1640914 grant to LL.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.