Reducing the capacitive opening between subwavelength metallic objects down to atomic scales or bridging the gap by a conductive path reveals new plasmonic spectral features, known as charge transfer plasmon (CTP). We review the origin, properties, and trending applications of this modes and show how they can be well-understood by classical electrodynamics and quantum mechanics principles. Particularly important is the excitation mechanisms and practical approaches of such a unique resonance in tailoring high-response and efficient extreme-subwavelength hybrid nanophotonic devices. While the quantum tunneling-induced CTP mode possesses the ability to turn on and off the charge transition by varying the intensity of an external light source, the excited CTP in conductively bridged plasmonic systems suffers from the lack of tunability. To address this, the integration of bulk plasmonic nanostructures with optothermally and optoelectronically controllable components has been introduced as promising techniques for developing multifunctional and high-performance CTP-resonant tools. Ultimate tunable plasmonic devices such as metamodulators and metafilters are thus in prospect. Plasmonics is a promising counterpart of nanophotonics which has witnessed major progresses in the control and manipulation of electromagnetic (EM) fields at extreme-subwavelength scales –3. CentrThere have been ongoing theoretical and experimental advancements to understand the possibility of direct dynamic charge transport between plasmonic objects in the absence of capacitive coupling between plasmonic particles. Relatively, researchers have shown the possibility of plasmon-driven charge transfer process in DNA-mediated metallic NP dimers , 34. In In spite of possessing interesting properties, CTPs are inherently suffering from lack of spectral tunability. Recent demonstrations that the functionality of CTP feature can be optimized by integrating bulk metallic systems with thermally and electronically controllable materials show why tunable CTP spectral features are important for implementing novel and advanced plasmonic tools , 43–46. In this review, we summarize recently introduced mechanisms that underpin the electron transition between plasmonic particles and trending applications of functional CTPs. One of the goals of this article is to present a comprehensive depiction of various modalities developed for the excitation of tunable CTPs. Besides, we explain the state-of-the-art approaches that have been proposed for enhancing the tunability of CTPs and the use of this spectral phenomenon in designing practical photonic tools such as modulators, switches, and nonlinear harmonic signal generators. Next, we illustrate how a careful integration of subwavelength plasmonic resonators with optically, thermally, and electronically functional materials leads to the emergence of multifunctional nanooptoelectronic and terahertz (THz) devices.via redistribution and transfer has been introduced as a simple approach to excite CTP spectral features and studied both theoretically and experimentally in nanoscale plasmonic systems  + Vxc[n(r)], where VH is the Hartree potential, n is the electronic density, Vxc is the exchange-correlation potential  to be able to have a proper electron spill-out. As plotted in VTLS = −4.6 eV. It is evident from z-axis): (a) the bare metallic dimer (gray line) and (b) the dimer with the TLS (red line). For the latter case, the background potential of the TLS is selected as equal to the NPs. In both cases, a bonding dipolar mode (BDP) [VTLS is varied to manipulate the position of the levels of the TLS. In this plot, each dot represents an energy level of the whole platform. Depending on the localization rate of the TLS levels, the size of the dot can be bigger, which is beneficial to differentiate localized levels in the TLS. A group of states are localized close to the Fermi level of the system, shown in black dashed line, for small numbers of VTLS. These states become more localized as the background potential is getting deeper and the states move to lower parts in the energy diagram. However, because of the strong localization, the interplay between the NPs is diminished. Particularly, when  VTLS is lower than -16.2 eV, an extra level starts to localize on the TLS. Lastly, in Figures VTLS values considered in In another recent study, Kulkarni and Manjavacas  investigximation ), n0 is de (BDP) , 76 appevia the direct transition of optically driven electrons across the bulk metallic paths between NPs suffer from the inherent lack of tunability. Recently, these challenges have effectively been addressed by using optoelectronically and optothermally tunable components in the purpose of the excitation of functional CTPs [As discussed in the previous section, in the quantum tunneling of energetic optically driven electrons, possessing an active control over the CTP spectral feature is limited to modifying the incident field intensity and/or morphological variations , 45. On nal CTPs –47, 77. sp2-hybridized carbon atoms, known as graphene, has received copious interest due to its significantly high electron mobility, mechanical flexibility, and exquisite optical properties [via modifying the generated carrier density [h is Planck's constant. Analogous to the plasmonic components, the spectral properties and plasmonic response of graphene sheet can be estimated by the Drude absorption model for a wide range of carrier densities [m is the electron mass and Γ is the transport scattering rate. The unprecedented levels of beam confinement and EM field enhancement by graphene allow for having electrostatic control over the plasmonic response in the absorption spectra [via adding small number of atoms. Using first-principle analyses, the plasmonic response of the entirely graphene-based dimer was studied for the intermediate junction with varying atomic row widths (4-8). The designed triangular structures forming the bowties are oriented with respect to the graphene lattice by assuming having armchair edges. This prevents the possible losses that can be naturally observed in zigzag-edge nanostructures, due to the presence of zero-energy electronic edge states [A one-atom thick layer of operties –82. Grapoperties , ultrafaoperties –87, nonloperties –90, and operties , 92. One density , 94. Thi density –95. This density –100. The density , 94(4)σensities , 94, 101ensities , 102(5) spectra . The suc spectra  demonstre states .3 atoms in each triangle), the researchers utilized the following settings: Fermi energy of the structures was to EF = 0.4 eV, the intrinsic damping as ħτ−1 = 1.6 meV, all corresponding to a DC mobility of 104 cm2/V·s [m = 0, 4, 8). It should be underlined that when m = 0, this means there is no atomically connection between proximal nanotriangles. Figures n). As shown in n resembles the number of carbon hexagons that are required to join the graphene triangles for m = 2. As can be explicitly seen in the extinction cross-section, by increasing the junction width, the spectral features show a trend from higher to lower energies. n. The size of the circles is prepared proportional to the intensity of the plasmon mode defined as the area under the corresponding plasmon peaks in the extinction profile. In the narrower junction limit, the spectra point out pronounced plasmonic features placed around ∼0.47 eV. On the other hand, when the junction becomes wider, the spectra are dramatically dominated by lower-energy plasmonic features around ∼0.22 eV. Finally, for the junction width intermediate geometries, there is a complex transition between the mentioned two regimes, with intermediate-energy features around ∼0.35 eV. As depicted in n) of the junction does not have a significant influence on the spectral changes. It is significant to note that the three distinguished behaviors of the plasmon energies declared before for all narrow, intermediate, and wide bridges perform for all sizes of the junction length.To begin with, Thongrattanasiri et al.  used the cm2/V·s .  Figuren) of the junction was fixed. Relatively, m = 0), validating a dipole-dipole interference that is not altered when a narrow junction (m = 2) is inserted between the two triangular resonators of the structure , one can see a distinct dipolar polarization pattern across the junction (see m = 8), similar to the fully metallic structures studied previously, the CTP becomes the dominant spectral response (see n) of the junction between neighbor graphene-based nanotriangles leads to effects that are consistent with the conclusions for the width of the bridge. Strictly speaking, Thongrattanasiri and teammates verified that the classical description of the investigated graphene nanostructures fails to regenerate the plasmonic behavior derived from first principles. This is obvious, for instance, for the junction width with intermediate sizes, the conventional computations oversight the intermediate-energy junction plasmons that have been observed in the quantum calculations. In addition, another disagreement between classical and quantum modes has been observed, in which the traditional approach predicted a smooth fadeout of the dipole-dipole mode exhibited by nonoverlapping triangles during insertion of the bridge, while the CTP shows a singular behavior, as it migrates towards zero energy in the limit of vanishing junction width.Further analyses help to understand the nature and properties of the plasmons in the graphene-based structures. In Figures ture see . Converstion see . In thisonse see . The sigIn T/T0 = 1 − 1/|1 + Z0σAC(ω)/(1 + ns)|2, where Z0 is the vacuum impedance, ns is the refractive index of the thin substrate, and the optical conductivity of graphene (σAC(ω)) can be taken by using the RPA principle. This leads to the strong capacitive coupling of plasmons at the charge neutrality point of graphene islands (resembling high resistance regime). This results in the excitation of a typical electric dipole. On the other hand, when the back-gate voltage is applied, the low resistance of graphene islands gives rise to the direct charge transition between the metallic resonators via the conductive atomic bridge and the excitation of THz-CTPs. 2/ITO substrate. Here, the ITO sublayer acts as a conductive surface for the applied voltages via the gate to control the AC photoconductivity of graphene islands. The resistance variations for graphene area in the presence of metallic objects are measured as a function of back-gate bias (Vbg), shown in Vbgcnp = 9.5 V). Moreover, the maximum resistance was measured around ~3.52 kΩ corresponding to the dielectric regime, while the lowest resistance was monitored for the graphene islands in the n-type phase correlating with the conductive regime. The geometrical parameters of the proposed unit cell are superimposed in the inset of this panel.To continue, we briefly consider the recent advances in enhancing the functionality of CTP resonances based on graphene-mediated metallic metastructures. Newly, Ahmadivand and colleagues have developed an approach to induce CTPs in gate-controlled graphene monolayer-integrated particle clusters in the THz spectra towards tailoring multifunctional metamodulators , where ħ is the reduced Planck's constant, vF is the Fermi velocity (106 m/s), and CA is the capacitance per unit area per charge of the multilayer substrate under the graphene islands. This enables active tuning of the frequency-dependent intraband photoconductivity of graphene by modifying the applied back-gate bias. Consequently, one can directly tune the conductance, resistance, and reactance of the graphene-enhanced bridge.To demonstrate the spectral properties of the device, firstly, the researchers computed and measured the spectral response under longitudinal polarized THz beam exposure at room temperature (300 K), as depicted in given by  EF = ħνF5 Hz, which led to a remarkable modulation depth up to 72% and fast operation speed with the rising and falling duration around 19 μs and 21 μs, respectively. From the modulation bandwidth principle, One other area that deserves special attention is that of optical modulation. Optical and optoelectronic metamodulators have previously been designed based on conventionally resonant structures such as Fano and EIT resonant metamolecules –117. The2Sb2Te5 (GST), have attained a copious interest for tailoring plasmonic and all-dielectric platforms from visible to infrared for various purposes, such as switching/modulation [As elaborated in Sections dulation , 122, sedulation , 124, andulation , 126, dudulation –130. In σGST(λ) = (c/2λ)(1 − εeff(λ)), where εeff(λ) is the effective permittivity of GST portion and c is the velocity of light in vacuum. Besides, depending on the crystallization level of the GST, the Lorentz-Lorenz effective-medium description can be performed using [εeff(λ) − 1)/(εeff(λ) + 2) = fc((εc(λ) − 1)/(εc(λ) + 2)) + fa((εa(λ) − 1)/(εa(λ) + 2)), where fi is the volume function of the ith phase as 0 ≤ fi = (ni/∑jnj) ≤ 1, where nj is the density of the jth phase. It is worth mentioning that to model the photothermal heating process, we utilized the multicapacitive cascading method [EH) in the platform can be defined as [EH = AQabsF(r), where Qabs is the numerical absorption coefficient, F(r) is the optical fluence of the impinging pulse, and A indicates the area of the dimer antenna (more comprehensive information can be obtained from the Supplementary Information file of the article). Furthermore, the normalized extinction spectra of the dimer system are presented for the following four different configurations of the bridge: full gold, air, amorphous GST (a-GST), and crystalline GST (c-GST)  is introduced to the monolithic gold nanowire, the charge transfer between the opposite sides of the system is hindered. Similar to the full gold case, a dipolar mode is appeared at higher energies, while a pronounced dipolar extreme is observed around 1.7 μm, owing to the strong capacitive coupling between gold nanorods. Moreover, when the gap area is filled up with GST , the dipolar peak around 1.7 μm is red-shifted to 2.2 μm with slightly increased intensity, due to negligible extinction coefficient (k ~ 0) of the a-GST in this regime [μm and its position is considerably red-shifted (δ ~ 100 nm) in comparison to the dipolar mode of a-GST, because of its absorptive behavior at low energies. It is significant to note that for all the studied conditions, the position and the amplitude of the induced dipolar mode shoulder are not affected by the changes made on the gold nanobridge. To verify this, the extinction profile of a bare gold dimer is plotted in E-field distributions are clearly demonstrated the presence of the CTP mode for the full gold structure. In the case of a-GST, opposite charges are accumulated around the dielectric region as well as in the surrounding gold nanodisks  88% of modulation depth at 1.55 μm, which is also known as the telecommunication band.In ed using –134  see . For coms regime . Once thidge see . Lastly,εeff(λ) of the GST sublayer for these calculations. Moreover, optically generated thermal power was quantified using the following equation: T = (AQaϕ/1.77ντ)/exp(−((t − t0)2/τ2)) [τ is the time constant of the incident light, ϕ is the beam fluence, r is the distance from the source, and t0 is the time delay. To verify the origin of the induced modes, the corresponding charge distribution profiles for the proposed device are investigated in In another recent study by Nooshnab and Ahmadivand , a novel0)2/τ2)) , where τIn recent years, the field of plasmonics has experienced rapid progresses in understanding the dynamics of conductively bridged and nearly touching metallic NPs. As we described in this focused review, many intriguing physical effects have been predicted in the systems supporting distinguished CTP excitations. So far, various theoretical and experimental investigations have been performed to realize oscillating electric current along the conductive junction, as a key principle of the CTP excitation, depending on the type of the nanosystem. However, related investigations have been limited to the excitation approaches and verifying this principle by standard measurements. Now that the CTP spectral feature has been reached in a broad range of platforms in modern nanophotonics, we believe that it is time to numerically and experimentally explore the modalities towards enhancing the functionality of this spectral phenomenon, eventually, to find its useful and practical applications. We also speculate that CTP states possess a strong potential to make a profound impact in the development of coming generation multifunctional nanophotonic instruments. More precisely, the transition between hybridized dipolar and distinct CTP modes have been explicitly demonstrated under capacitive and conductive coupling regimes, respectively. Highly attractive features of CTP resonances have opened the door for novel active devices and applications, including but not limited to surface-enhanced IR absorption (SEIRA), surface-enhanced Raman scattering (SERS), optical switching, modulation, and waveguiding; however, active manipulation of these resonances using functional materials is overlooked.In this review, we briefly presented the recent accomplishments in the use of versatile materials towards actively tunable CTP-based nanoscale tools. As indicated in the considered studies, having an active control on the CTP mode is possible using functional materials like graphene and GST, rather than applying morphological variations. The exceptional optical and electrical characteristics of these materials have allowed researchers to design active CTP-resonant devices. We envisage that this review will provide detailed understanding for the evolution from passive to active CTP-based platforms and pave the road for developing next-generation nanophotonic devices to reach new functionalities.


Bacillus subtilis with increased competitiveness towards other microorganisms. In particular, nonribosomal peptides (NRPs) have an enormous antimicrobial potential by causing cell lysis, perforation of fungal membranes, enzyme inhibition, or disruption of bacterial protein synthesis. This knowledge was primarily acquired in vitro when B. subtilis was competing with other microbial monocultures. However, our understanding of the true ecological role of these small molecules is limited. In this study, we have established soil-derived semisynthetic mock communities containing 13 main genera and supplemented them with B. subtilis P5_B1 WT, the NRP-deficient strain sfp, or single-NRP mutants incapable of producing surfactin, plipastatin, or bacillaene. Through 16S amplicon sequencing, it was revealed that the invasion of NRP-producing B. subtilis strains had no major impact on the bacterial communities. Still, the abundance of the two genera Lysinibacillus and Viridibacillus was reduced. Interestingly, this effect was diminished in communities supplemented with the NRP-deficient strain. Growth profiling of Lysinibacillus fusiformis M5 exposed to either spent media of the B. subtilis strains or pure surfactin indicated the sensitivity of this strain towards the biosurfactant surfactin. Our study provides a more in-depth insight into the influence of B. subtilis NRPs on semisynthetic bacterial communities and helps to understand their ecological role.Secondary metabolites provide  In nature, bacteria live in complex communities where they interact with various other microorganisms. Most microbial communities are influencing biochemical cycles and impact agriculture, from which the latter is primarily mediated due to plant-growth promotion –4. ExtenSecondary metabolites (SMs) are believed to be important mediators of the interactions between microorganisms . Many ofBacillus subtilis is a well-studied soil bacterium and is used as a model organism for biofilm formation and sporulation [B. subtilis species complex have exceptional plant growth promoting and plant health improving properties by suppressing plant pathogenic bacteria and fungi [Bacillus spp. affect the indigenous microbial communities. Gadhave et al. have shown that the supplementation of B. subtilis, Bacillus amyloliquefaciens (now identified as Bacillus velezensis), and Bacillus cereus to the roots of broccoli plants led to species-dependent changes in the diversity, evenness, and relative abundances of endophytic bacterial communities [B. subtilis and other Bacillus spp. produce various SMs [B. subtilis NRPs, the phosphopantetheinyl transferase Sfp is needed since it has been shown to activate the peptidyl carrier protein domains, converting it from the inactive apo-form to the active holo-form [B. subtilis has four sfp-dependent SMs, of which three are synthesised by NRPS gene clusters  and one by a hybrid NRPS–PKS gene cluster -based growth monitoring of the selected strain Lysinibacillus fusiformis M5 confirmed the impact of B. subtilis-produced surfactin on the growth.In this study, we focus on soil-derived semisynthetic bacterial mock communities and describe how these are affected by a libactin . It was B. subtilis WT P5_B1, the corresponding NRP-deficient strain sfp, or the single-NRP mutants srfAC, ∆ppsC, and ∆pksL, respectively, incapable of producing either surfactin, plipastatin, or bacillaene, and kept the untreated culture as a control , Candidatus Udaeobacter (7–10%), and Gaiella (3–4%). The communities of the 12 h precultivated soil suspension consisted primarily of the two genera Bacillus (56–65%) and Acinetobacter (29–34%). Additional genera with an abundance higher than 1% were Lysinibacillus (1.2–3.2%), Pseudomonas (1.0–2.2%), and Viridibacillus (0.6–1.5%). The genus richness of the four precultured soil suspensions was between 12 and 18 of the total 21 genera.We established soil-derived semisynthetic mock communities and supplemented them with  control . To inveeplicate . We inveDiversity analyses were performed to determine the overall impact of the NRPs on the diversity of the bacterial mock communities. The read numbers varied among the different samples  amplicon sequence variants (ASVs) to the nMDS ordination and plotted them as vectors to investigate the differences between the mock communities. The analysis indicated that three ASVs, taxonomically assigned to the genera Lysinibacillus, Acinetobacter, and Viridibacillus, correlated with the control and two sfp-treated communities. This observation suggests that the absence of NRP-producing B. subtilis resulted in an increased abundance of these. Furthermore, two ASVs of the genus Acinetobacter correlated best with the communities supplemented with the NRP-producing B. subtilis strains, hinting a higher frequency of these in NRP-treated communities. Additionally, three ASVs, identified as Pseudomonas, Citrobacter, and Sphingobacterium, correlated with two communities treated with the surfactin mutant. A similar but smaller correlation with two bacillaene mutant-treated communities was detectable as well. These results imply a negative impact of either surfactin or bacillaene on the four ASVs. Interestingly, the vector-based analysis suggests that, depending on the ASVs, the genus Acinetobacter is both positively and negatively affected by the NRPs.Therefore, we determined the beta diversity only for the treated and untreated mock communities cocultivated for 48 h. The analysis underlined a high similarity in the composition of the mock communities treated with  strains . HoweverB. subtilis strains on the diversity. However, the beta diversity results suggested that the addition of NRP-producing B. subtilis strains influenced the composition of the mock communities. Mainly ASVs belonging to the genera Lysinibacillus, Viridibacillus, and Acinetobacter were affected by the presence or absence of B. subtilis NRPs in the bacterial mock communities.In conclusion, the alpha diversity analyses revealed that species richness and evenness were reduced in the in vitro communities compared to the soil community. Furthermore, 12 h preincubated soil suspensions showed a reduced diversity compared to the mock communities incubated for 48 h. Nevertheless, we could not detect an effect of the supplemented B. subtilis-treated or untreated mock community were Acinetobacter, Lysinibacillus, Pseudomonas, Chryseobacterium, Bacillus, Sphingobacterium, Stenotrophomonas, Paenibacillus, Citrobacter, Serratia, Achromobacter, Viridibacillus, and Pantoea. Noteworthy, the prevalence of the Bacillus genus was comparable in the B. subtilis-treated communities (4–9%) and the control (5–10%). In the latter, the present Bacillus ssp. originated only from the soil suspension, highlighting that the additional supplementation of B. subtilis did not affect the relative abundance of the genus Bacillus after 48 h cocultivation. Interestingly, the only genera detected in both the in vitro mock communities and the soil samples were Bacillus, Lysinibacillus, and Paenibacillus. The remaining most abundant genera in the mock communities were below the detection limit.The diversity, in particular the species evenness, increased independently of the treatment in all established mock communities, compared to the precultivated soil suspensions and contained 11–18 genera . The mosB. subtilis WT-treated communities revealed that Lysinibacillus and Viridibacillus were significantly decreased 9.4-fold (P ≤ 0.001) and 8.3-fold (P ≤ 0.01), respectively, in the communities supplemented with B. subtilis WT  of Lysinibacillus in the sfp-treated communities compared to the untreated communities, and thus a greatly diminished effect compared to the WT-treated samples was evident  significantly reduced in these communities. The direct comparison of WT- and sfp-treated communities confirmed the NRP-dependent suppression of both Lysinibacillus and Viridibacillus in the WT-treated communities and the suppression of Stenotrophomonas in the sfp-treated communities , which was significantly different compared to all other B. subtilis-treated assays  of Lysinibacillus (1.2%) compared to the control communities was discovered. Furthermore, when we added the NRP-deficient strain sfp, we could notice a significantly higher abundance of Lysinibacillus (8.6%) compared to the WT-treated communities (P ≤ 0.001) but still a significantly lower prevalence compared to the control communities (P ≤ 0.05). Compared to the WT-treated communities, the frequency of Lysinibacillus was slightly but not significantly higher in the communities treated with the single-NRP mutants srfAC (2.0%) and ∆ppsC (3.3%). The abundance of Lysinibacillus in the assays containing the ∆pksL strain (5.3%) was significantly higher (P ≤ 0.01) than in the WT-treated assays. However, the Lysinibacillus abundance in ∆pksL-treated communities was not significantly different from the ∆ppsC- or sfp-treated communities. In summary, Lysinibacillus was affected by the addition of B. subtilis independent of the NRPs, but when B. subtilis strains capable of producing them were present, the impact on Lysinibacillus was enhanced. Furthermore, the results indicate that bacillaene had the strongest and surfactin the weakest effect on Lysinibacillus in the mock communities.Concentrating on d assays . HoweverB. subtilis was Viridibacillus, which had a very low abundance in the control mock communities (0.49%) compared to Lysinibacillus  abundance (0.03%) compared to the control communities. Notably, in two of the WT-treated community replicates, Viridibacillus was below the detection level. Nevertheless, the abundance of this genus in the sfp-treated communities (0.26%) was statistically not significant in comparison to the WT and the control communities. Furthermore, the addition of the single-NRP mutants srfAC, ∆ppsC, and ∆pksL resulted in communities with Viridibacillus frequencies similar to the WT-treated communities . Viridibacillus as well as Lysinibacillus was affected by the addition of B. subtilis to the communities. However, no particular NRP could be assigned to the reduced frequency of Viridibacillus.The second genus affected by the addition of Lysinibacillus was negatively affected by the addition of B. subtilis P5_B1 WT and that NRPs enhance the suppression. To dissect the direct impact of a particular NRP in this inhibition, we monitored the growth of L. fusiformis M5, a previously isolated Lysinibacillus species [B. subtilis WT and the corresponding NRP mutants  ,64. 1 g Environmental- and semisynthetic-community genomic DNA was extracted from either 250 mg soil or 250 µL bacterial culture, respectively, by using the DNeasy PowerSoil Pro Kit (QIAGEN) and following the manufacturer’s instructions.The V3-V4 region of the 16S rRNA gene was PCR-amplified from the extracted DNA samples using Fw_V3V4 (5’-CCTACGGGNGGCWGCAG-3’) and Rv_V3V4 (5’-GACTACHVGGGTATCTAATCC-3’) primers that were tagged with short barcodes with a length of eight nucleotides, listed in Table S2, The multiplexed sequencing data was imported into the QIIME 2 pipeline (version 2020.6) –66. The B. subtilis WT as well as in the communities supplemented with B. subtilis sfp, were investigated by calculating the abundance ratios of the different treated communities for each replicate. If species were not detected in some of the replicates, 0 values were replaced with the lowest detected value of the genus to avoid infinite values or 0 values in the ratio calculations. Rarefaction curves of the samples were calculated and visualised with the R package ranacapa [B. subtilis-treated and untreated samples were performed with ASV counts multiplied by factor 100,000 and transformed into integer proportions. The alpha diversity was estimated with the Shannon diversity index in the R package phyloseq [R2 > 0.6) ASVs were added to the nMDS ordination plot. All graphical visualisations were realised with ggplot2 [QIIME 2 artefacts were imported into the R software (4.0.2) with the R package qiime2R, and further analyses were conducted in the R package phyloseq –76. The ranacapa . Diversiphyloseq . The cor ggplot2 .The statistical significance was determined with the square roots of the tested values. The normality and equality of the variances were tested with the Shapiro–Wilk normality test and the Levene test, respectively. If one of the tests was rejected, the nonparametric Kruskal–Wallis rank sum test was performed instead. The statistical significance of pairs was determined with the Welch two-sample t-test, and the differences among groups >2 was determined with the one-way analysis of variance (ANOVA) test and the Tukey HSD test. The statistical significance was determined with an alpha level <0.05.B. subtilis strains were harvested from cultures grown in TSB medium at 37 °C and 250 rpm for 48 h immediately before the growth experiments. The cultures were adjusted to OD600 3.0 and centrifuged for 4 min at 5,000g. Subsequently, the supernatants were passed through 0.22 µm filters and stored at 4 °C. The growth experiments were performed in 96-well microplates. The wells of the first column were filled with 30 µL 10 × TSB, 30 µL L. fusiformis culture adjusted to OD600 0.1 in 1 × TSB, and 240 µL of the appropriate spent B. subtilis medium or water (untreated control). 100 µL L. fusiformis culture adjusted to OD600 0.01 in 1 × TSB was added to the wells of the remaining columns. A 1.5-fold serial dilution of the spent media was performed column-by-column. A surfactin stock solution was prepared by dissolving 10 mg of surfactin (Sigma-Aldrich) in 1 mL methanol (MeOH). The wells of the first column were filled with 170 µL 1 × TSB, 20 µL L. fusiformis culture adjusted to OD600 0.1 in 1 × TSB, and 10 µL surfactin, 10 µL MeOH (solvent control), or 10 µL 1 × TSB (untreated control). To the wells of the remaining columns, 100 µL L. fusiformis culture was added adjusted to OD600 0.01 in 1 × TSB. A 2-fold serial dilution of surfactin or MeOH was performed column-by-column. In both assays, the growth of L. fusiformis was monitored in a microplate reader (BioTek Synergy HTX Multi-Mode Microplate Reader). The microplates were incubated at 30 °C with continuous shaking , and the OD600 was measured in 15 min intervals over 24 h. All graphical visualisations were prepared using ggplot2 [Spent media of  ggplot2 .File 1Bacterial strains used in this study, 16S rRNA V3-V4 primer list, number of sequencing reads per sample, and supporting figures.


Aptamer-immobilized graphene field-effect transistors (GFETs) have become a well-known detection platform in the field of biosensing with various biomarkers such as proteins, bacteria, virus, as well as chemicals. A conventional aptamer immobilization technique on graphene involves a two-step crosslinking process. In the first step, a pyrene derivative is anchored onto the surface of graphene and, in the second step, an amine-terminated aptamer is crosslinked to the pyrene backbone with EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide) chemistry. However, this process often requires the use of organic solvents such as dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) which are typically polar aprotic solvents and hence dissolves both polar and nonpolar compounds. The use of such solvents can be especially problematic in the fabrication of lab-on-a-chip or point-of-care diagnostic platforms as they can attack vulnerable materials such as polymers, passivation layers and microfluidic tubing leading to device damage and fluid leakage. To remedy such challenges, in this work, we demonstrate the use of pyrene-tagged DNA aptamers (PTDA) for performing a one-step aptamer immobilization technique to implement a GFET-based biosensor for the detection of Interleukin-6 (IL-6) protein biomarker. In this approach, the aptamer terminal is pre-tagged with a pyrene group which becomes soluble in aqueous solution. This obviates the need for using organic solvents, thereby enhancing the device integrity. In addition, an external electric field is applied during the functionalization step to increase the efficiency of aptamer immobilization and hence improved coverage and density. The results from this work could potentially open up new avenues for the use of GFET-based BioMEMS platforms by broadening the choice of materials used for device fabrication and integration. DS) of the GFET. A significant difference between GFETs and other conventional FETs is their ambipolar transfer (IDS-VGS) curve which results in a minimum channel current at the Dirac voltage also known as the charge neutrality point (VCNP). Any change in the electric field induced by the adsorption of the charged molecules at the graphene surface essentially causes a shift in the charge neutrality point enabling GFETs to be used for ultra-sensitive detection of the target biomolecules [In recent years, graphene-based field-effect transistors (GFETs) and their uses as sensing platforms have been greatly successful in developing various microfluidic and lab-on-a-chip-based biosensors ,2. Theirolecules ,6. To enhance the target selectivity in analyte detection, GFETs are commonly integrated with bioreceptors such as aptamers, antibodies and so forth. In this work, aptamers are used as representative target recognition probes. A major part of the implementation of such aptameric GFET devices is the reliable immobilization of aptamer probes onto the graphene channel of the device. In general, there are two main strategies for functionalizing aptamers on graphene, namely, the covalent and the non-covalent immobilization approaches. While the covalent approaches offer certain advantages over non-covalent methods in terms of stability and functionality, they unavoidably altar the physical properties of graphene. Hence, non-covalent modifications have been frequently used in order to maintain the inherent properties of a pristine graphene . A typicE. Coli with the aid of pyrene-tagged DNA aptamers [A commonly used pyrene-based crosslinker, such as PBASE (1-pyrenebutyric acid N-hydroxysuccinimide ester) as shown in aptamers . In theiaptamers . Howeveraptamers , the maiaptamers ,17. D = 5.4 nM) and the resuspension buffer were purchased from Base Pair Biotechnologies, Inc. . The aptamer’s affinity has been thoroughly characterized by the manufacturer and is shown to be specific toward IL-6 proteins. The predicted secondary structure of the aptamer sequence is presented in 2/Si was purchased from Graphenea, Inc. . The gold electrodes (source/drain) surrounding the graphene channel were passivated with insulating layers consisting of Al2O3 (50 nm)/Si3N4 (100 nm). A polydimethylsiloxane (PDMS) well (3 mm diameter) was then integrated in-house to contain the liquid gate on the passivated area of the electrode as well as to reduce liquid evaporation during measurements and incubation steps. The aminated aptamers having the specific affinity to mouse IL-6  was loaded into the well and a negative electric field (−400 mV) was applied to the solution using a wire inserted into the PTDA solution for 4 h as shown in 2 were exposed to the sensing area (PDMS well) for 10 min each. To investigate the selectivity and specificity, 100 nM of lysozyme (Lys) protein was also exposed to the IL-6 aptamer modified GFET in the same buffer for the same duration. After functionalizing the GFETs with IL-6 binding aptamers, various concentrations  of IL-6 protein in 0.01× PBS with 2 mM MgClD–VGS transfer characteristics. Voltage control and data acquisition were performed using a LabVIEW program. During the measurement, the drain–source voltage (VDS) was biased at 100 mV and the drain current (ID) was read while the liquid–gate voltage (VGS) was linearly scanned from 0 V to 1 V with a voltage step of 12.5 mV using the in-plane gold gate electrode. A scan rate of 12.5 mV/s was maintained so that ID is stabilized to ensure reliable measurements of the ID–VGS transfer curves. For electrical measurements, the devices were placed on a probe station  and a Keysight B2902A source/measure unit (SMU) was used to measure the ICNP) or the Dirac voltage (VDirac) [The transfer characteristics of a transistor in a linear region can be described as follows :IDS=WL.C(VDirac) . The sloessed as :gm=dIDSd2/V/s and 2/V/s, where 2 [Therefore, the average carrier mobilities for holes and electrons were calculated to be: 5 µF/cm2 . This hiON/IOFF) of the drain-source current. The larger the value of the on-off ratio, the better the sensitivity of the GFET since the device will exhibit better immunity to noise. An on-off ratio value of ~8 was calculated for the used devices which is above average for GFET devices grown by a CVD technique [Another important parameter that affects the sensitivity of the GFET devices is the on-off ratio (Iechnique . CNP) for the 6 GFET devices on a single chip. As seen in the figure, Ag/AgCl gate electrode gives the lowest VCNP among the three gate electrodes. However, the gold electrode provides the lowest device-to-device variations among the devices on a single chip. We also investigated the effects of different gate materials  on the GFET transfer characteristics illustrated in GS) calculated from the IGS-VGS curves  has been plotted in Although Ag/AgCl demonstrates the best performance in terms of the operating voltage, gate leakage and gate hysteresis, in-plane gold electrode was used throughout the experiments as it gives the highest uniformity of the charge neutrality point among the devices on a single chip. Moreover, the in-plane configuration of the gold electrode which can be fabricated at the same lithography step as the source and drain electrodes enhances the compactness of the setup and allows the potential integration with the microfluidic platform . −1 (C=O stretching in the amide I) and the broad stretching vibration peak around 3300–3550 cm−1  confirm the amide bond formation [The synthesized PTDAs were first characterized to verify the presence of an amide bond between the PBASE and the aminated aptamer. The amide bond was characterized using the Fourier-transform infrared (FTIR) spectroscopy as presented in −1 indicates the anchoring of the PTDAs by π-π stacking interaction between the pyrene ring of the PTDA and the basal plane of graphene [After the amide bond was confirmed by the FTIR spectroscopy, the PTDAs were immobilized on bare graphene. The presence of the PTDAs on graphene was confirmed by Raman spectroscopy. For Raman measurements, two samples were prepared, a blank graphene and a graphene functionalized with PTDAs. The Raman spectra for the PTDA-modified graphene were taken at 3 different spots on the surface. graphene .DS-VGS curves. CNP) compared to the case without the external electric field. With a negative potential at the gate, the negatively charged single-stranded PTDAs tend to migrate towards the graphene surface due to the electrostatic repulsion resulting in an increased density of the immobilized PTDA probes [CNP with respect to the blank GFET device for the two cases. With the electric field applied, the negative shift in the charge neutrality point was measured to be 123.53 mV which is approximately 2.5 times larger than that without the electric field.The anchoring of the PTDAs on graphene surface was also verified electrically by measuring the GFET transfer curves before and after the probe attachment. As shown in A probes . The bar2 for 10 min. Once the selectivity of the IL-6 binding aptamers was confirmed, the sensor was then exposed to various concentrations  of the target protein (IL-6) in the same buffer and for the same exposure time. After the successful functionalization of the GFET with the PTDAs, the device was exposed to IL-6 proteins to characterize its sensing performances. Prior to IL-6 exposure, in order to test the sensor’s selectivity to its target, the GFET-based IL-6 sensor was exposed to 100 nM of lysozyme protein (a model interfering species) in 0.01× PBS with 2 mM MgClAlso, the specificity of sensor was examined by functionalizing the GFET with a random sequence aptamer using the same protocol as the IL-6 aptamer and exposing different concentrations of IL-6 protein. The results are presented in CNP) using the formula ΔVCNP/ΔVCNP,max, where VCNP,max is the charge neutrality point corresponding to the maximum IL-6 concentration tested. With this detection method, the limit of detection of 100 pM was achieved. Although this detection limit is relatively higher than that achieved by the standard two-step immobilization of aptamers [2) but with the pH adjusted to ~3.64.aptamers , the benaptamers . As an eD is estimated to be 3.4 nM, similar to the value (5.4 nM) reported by the manufacturer for the aptamer-target pair. The limit of detection (LOD) of the GFET-based IL-6 sensor under this pH environment was calculated to be ~8 pM which is an order of magnitude larger than that under the physiological pH (pH ~7.4). We have developed a facile and rapid immobilization technique to attach target recognition probes on the GFET-based biosensing platform. The developed sensor was able to selectively measure IL-6 protein biomarker with the detection limit in the picomolar range. The sensitivity can be further improved by increasing the incubation time, purification steps as well as by adjusting the buffer pH to an acidic region. The proposed organic solvent-free aptamer immobilization technique is not only polymer friendly  but also simplifies and shortens the graphene modification process by eliminating the extra step needed for anchoring the linker molecules and the subsequent washing steps. We have also demonstrated that an external electric field can be used to enhance the efficiency (~2.5 times) of the aptamer immobilization on the graphene surface. Our proposed technology has the potential to be used in monitoring IL-6 from real physiologically relevant fluid samples such as sweat, serum and cerebrospinal fluid.


In our case, the NGr-1 sample which has the lowest doping level and the highest concentration of pyrrolic-N among all nitrogen-doped samples exhibits the best electrochemical parameters: a very small Rct (38.3 Ω), a large Kapp (13.9 × 10−2 cm/s) and the best electrochemical response towards 8-hydroxy-2′-deoxyguanosine detection (8-OHdG).Three nitrogen-doped graphene samples were synthesized by the hydrothermal method using urea as doping/reducing agent for graphene oxide (GO), previously dispersed in water. The mixture was poured into an autoclave and placed in the oven at 160 °C for 3, 8 and 12 h. The samples were correspondingly denoted NGr-1, NGr-2 and NGr-3. The effect of the reaction time on the morphology, structure and electrochemical properties of the resulting materials was thoroughly investigated using scanning electron microscopy (SEM) Raman spectroscopy, X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), elemental analysis, Cyclic Voltammetry (CV) and electrochemical impedance spectroscopy (EIS). For NGr-1 and NGr-2, the nitrogen concentration obtained from elemental analysis was around 6.36 wt%. In the case of NGr-3, a slightly higher concentration of 6.85 wt% was obtained. The electrochemical studies performed with NGr modified electrodes proved that the charge-transfer resistance (R Graphene, a single layer of graphite, possesses various interesting properties such as large surface area, high thermal and electrical conductivity and good optical transparency ,2,3. Curct) and large apparent heterogeneous electron transfer rate constant (Kapp). In addition, we originally proved the relationship between these two parameters and the electro-catalytic effect of N-doped graphene towards the detection of cancer biomarker, 8-hydroxy-2’-deoxyguanosine (8-OHdG).In this paper, we present for the first time the influence of the reaction time on the morphology, structure and electrochemical properties of nitrogen-doped graphenes. We show that the hydrothermal reaction may produce high quality nitrogen -doped graphene with very low charge-transfer resistance (R4[Fe(CN)6] and potassium chloride (KCl) were purchased from Merck (Germany). N,N-dimethylformamide (DMF) was bought from Sigma-Aldrich (Germany). 8-hydroxy-2’-deoxyguanosine (8-OHdG) was purchased from the Cayman Chemical Company. For the synthesis of nitrogen-doped graphene the following chemicals were used: graphene oxide, prepared using a modified Hummers method as previously reported  . In contrast, for the bare GC electrode the two waves were very broad and the peak potential separation large (400 mV)  in the presence of a redox indicator, K(400 mV) b. GO mod(400 mV) c.peak) vs. ʋ1/2 plot  (0.2 M KCl supporting electrolyte) at a bias corresponding to the peak potential of each electrode  within 0.1–106 Hz frequency range.Next, the interfacial properties of bare and N-doped graphene modified electrodes were evaluated using electrochemical impedance spectroscopy. The spectra were recorded in 10ct), and a straight line at low frequencies, characteristic to the diffusion of the electro-active species towards the electrode/solution interface (Warburg impedance). In the case of the GC/GO electrode, the semicircle is highly extended due to the insulating properties of graphene oxide. In contrast, for the electrodes modified with nitrogen-doped graphene the impedance values decreased by one order of magnitude, both the semicircle and the diffusive line (see the inset in In s), the charge-transfer resistance at the electrode/solution interface (Rct), the Warburg impedance due to the diffusion of ions in solution (ZW) and a constant phase element (CPE) that models a non-ideal capacitor.For the bare GC electrode, the best fit was obtained with a circuit containing the solution resistance , the surface is significantly different from that of bare GC. The successive GO layers deposited on top of the substrate were forming a porous structure where the insertion of ions from solution was hindered by the surface charges. Therefore, the equivalent circuit was a more complex one and contained the solution resistance, two RC parallel circuits (where the capacitances were replaced by CPEs) and the Warburg impedance . One of s, Rct and CPE1; In the case of electrodes modified with nitrogen-doped graphene, the surface is less hydrophilic but still has increased roughness compared with bare GC, and therefore, the best fit was obtained when the Warburg impedance was replaced with CPE, while the other circuit elements were preserved 6]3−, may easily transfer the electrons. The determined Rct values are indeed significantly lower for all GC/NGr electrodes, in comparison with the bare GC or GC/GO electrode. This is also in excellent agreement with the previous electrochemical measurements, where the CVs recorded with the GC/NGr electrodes exhibits the characteristics of a reversible redox process . According to the literature ,60, the cess see .app) calculated for each electrode (Equation (6)) [n is the number of electrons transferred during the redox reaction (n = 1); A is the active area of the electrode (cm2); Rct is the charge-transfer resistance obtained from the fitted Nyquist plots (Ω); C is the concentration of the redox species (mol/cm3).The value of apparent heterogeneous electron transfer rate constant (Kion (6))  gives al−4 cm/s) but is considerably higher for all NGr-modified electrodes (6.55−13.9 × 10−2 cm/s). The largest value was that corresponding to NGr-1 material, which had the lowest doping level and the highest concentration of pyrrolic-N, among all nitrogen-doped graphene.Its value has the same order of magnitude for GC/GO and bare GC (2.1−2.6 × 10c), which reflect the charge-storage capability of the synthesized materials. Hence, the graphene samples with the lowest doping level (GC/NGr-1 and GC/NGr-2) have the largest capacitive current, of 1.15 × 10−7 A and 1.01 × 10−7 A, respectively. In comparison, all the other samples have lower values, such as 5.58 × 10−8 A (GC/NGr-3), 1.48 × 10−8 A (GC/GO) and 2.39 × 10−8 A  and the results are shown in −7−10−3 M) in pH6 PBS supporting electrolyte . It is followed by GC/NGr-3, GC/NGr-2 and bare GC. The poorest electrochemical signal was obtained with the GC/GO electrode, due to the insulating properties of graphene oxide. The electrochemical parameters obtained from the calibration plots  for bare and graphene-modified electrodes are listed in Next, linear sweep voltammograms were recorded with each electrode in solutions containing various concentrations of 8-OHdG (10a)(L was obtained for GO (17.11 nm), followed by the NGr-1 (16.24 nm) and NGr-2 (16.16 nm) samples, which according to elemental analysis had similar amounts of nitrogen (6.36 wt%). For NGr-3 sample, the La value is slightly lower (16.12 nm), being in good agreement with the higher doping level (6.85 wt%). In addition, the EIS studies conducted with NGr-modified electrodes indicated that the sample with the lowest doping level and the highest concentration of pyrrolic-N among all nitrogen-doped graphene (NGr-1) exhibits the best electrochemical parameters: a very small Rct (38.3 Ω), a large Kapp (13.9 × 10−2 cm/s) and a low detection limit (9 × 10−8 M) for 8-OHdG.Nitrogen-doped graphenes with various concentrations of heteroatom  were prepared using the hydrothermal method with urea as reducing/doping source for graphene oxide. The structural characterization of the synthesized materials revealed that after doping, significant changes occurred in the XRD, Raman and XPS spectra, confirming the presence of heteroatom. For exemplification, the largest value for the in-plane crystallite size


It has been observed that the Dirac cones can be easily shifted along the symmetry lines without breaking the degeneracy. We have attained two different conditions based on the sole relations of hopping parameters and on-site energies to break the degeneracy. Further, in order to perceive the topological aspect of the system we have obtained the phase diagram and Chern number of Haldane model. This exact analytical method along with the supported DFT computation will be very effective in studying the intrinsic behaviour of the Dirac materials other than graphene.Present work reports an elegant method to address the emergence of two Dirac cones in a non-hexagonal graphene allotrope S-graphene (SG). We have availed nearest neighbour tight binding (NNTB) model to validate the existence of two Dirac cones reported from density functional theory (DFT) computations. Besides, the real space renormalization group (RSRG) scheme clearly reveals the key reason behind the emergence of two Dirac cones associated with the given topology. Furthermore, the robustness of these Dirac cones has been explored in terms of hopping parameters. As an important note, the Fermi velocity of the SG system ( In particular, the exceptionally high carrier mobility of graphene lies in the fact that the dynamics of charge carriers is governed by the Dirac equation3. Furthermore, these gapless Dirac cones in graphene are robust under some of the external perturbations viz. structural deformation or chemical functionalizations5. However, the only dispute that restrict the potential application of graphene in modern opto-electronic devices is its zero band gap nature7. Afterwards, a plethora of investigations have been performed on graphene in order to tune its band structure10. It is to note that other two dimensional (2D) hexagonal systems i.e. silicene and germanene11 also exhibit linear dispersion relation near Fermi level (EF). Previously, it was the perception that honeycomb lattice is necessary for the existence of Dirac cones. However, modern scientific advances have discarded the above mentioned fact13. Alternatively, Wang et al.14 have critically explored the conditions for the emergence of Dirac cones in 2D lattice. It is to note that the rectangular lattices viz. buckled T-graphene13, 6,6,12-, 14,14,14-, and 14,14,18-graphyne15 also satisfies the condition14. Furthermore, Xu et al.16 have availed structural search method in combination with first-principles calculations to introduce three tetragonal graphene allotropes S-, D- and E-graphene. These allotropes are sp2 hybridized and dynamically stable. Surprisingly, the linear valence and conduction bands in S-graphene touchs each other at two distinct Dirac points in the irreducible Brillouin zone (IBZ). Whereas, D- and E-graphene exhibit only one Dirac point similar to graphene. Therefore, in this work we want to address the key reason behind the occurrence of two Dirac cones in S-graphene. In addition, we are also interested in examining the robustness of the Dirac cones to tune the band structure of S-graphene.The first experimental isolation of graphene17. This phase of materials exhibits the quantized Hall resistance in case of the integer quantum Hall effect (IQHE)19. The IQHE can not be explained in terms of the spontaneous symmetry breaking of Landau theory20. It has been explained with the help of topological band theory. For the purpose, Haldane21 explicitly introduced a scheme to break the time-reversal symmetry giving rise to the anomalous quantum Hall effect (AQHE). The time reversal symmetry can be broken with the help of the staggered magnetic flux in a way that the net magnetic flux in each plaquette is invariably zero. The translational symmetry of the lattice has thus been restored. These systems are better known as topological Chern insulators. It is to note that the Chern insulators exhibit non-zero Hall conductance in the absence of any global magnetic flux22. The topological phase transition connecting two regimes with a distinct Chern numbers (0 to ±1). The Chern number is a topological index that reveal the information about the phase evolution of wavefunctions around the Brillouin zone (BZ) torus. Moreover, remarkable experimental advances in the field of ultracold atoms26 and photonic systems30 have successfully evinced the realization of non-trivial topological phases. One of the key advantages of Haldane model is certainly the experimental accessibility of its phase diagram in case of the non-interacting ultracold systems25. Besides, the lattice dynamics can be efficiently attainable via manipulating any single site31. Furthermore, an extended version of Haldane model32 exhibits that the long range interaction generates additional satellite Dirac points. These Dirac points play a crucial role in explaining the sudden quenching dynamics of the model. The nonequilibrium dynamics of the edge current of the semiopen Haldane model has also been reported33. Moreover, these symmetry protected edge states are extremely important in the study of the superconductors with Majorana Fermions34, spin Hall insulators37, three dimensional topological insulators38 etc. The paper is organized as follows. In the next section (section-II) we have discussed the exact RSRG scheme on S-Graphene like tetragonal lattice. In section-III we have described an analytical description on the emergence of two Dirac cones in the IBZ of the S-graphene. In the subsequent section-IV the robustness of the Dirac cones has been well explored. In addition, we have proposed a method in section-V to obtain the Haldane lattice of S-graphene for describing its topological phase with non-zero Chern number. Finally, the conclusion has been drawn at the end of this paper.The topological insulators are the bulk insulators with conducting surfaceset al.16 as a stable allotrope of graphene called S-graphene. The tetragonal unit cell of S-graphene is defined by the lattice vectors 42 to compute the dispersion (E-k) relation associated with the system through tight binding Hamiltonian. In particular, we have decimated out the preferred subset of atomic sites of the original lattice to achieve a scaled version of it. The scaled lattice, however, carries all the information of the original lattice. We can perform the process several times without losing any generality. The RSRG process has been initiated by evaluating the set of difference equations for the original lattice using Eq. Our primary aim is to address the emergence of two Dirac cones and their robustness associated with the lattice shown in Fig. i-th and the j-th sites. The electron’s hopping is restricted between nearest neighbour (NN) only. It is clear from Fig. Here, F = Now, we want to decimate the red shaded atoms shown in Fig. F. In a similar vein, we have repeated the RSRG scheme for the output lattice of the first RSRG process in a more rigorous manner. In accord with the first RSRG scheme, we have chosen another section as shown in Fig. As a consequence, we are left with a new scaled lattice, depicted in Fig. As an example, we have decimated the atomic sites b and e using the following relationsc and f site also for the corresponding decimation and the renormalized lattice is depicted in Fig. We can use similar relations for t″ present in the system. Such a two-band structure can generically be treated as a two-level system that corresponds to a two-dimensional Hilbert space  have the same value similar to the original lattice. Therefore, the dispersion relation has the formWe now start with the most trivial case, i.e. all the hopping parameters of the original lattice are uniform say, E-k spectra over the entire tetragonal BZ. As already anticipated we have observed the occurrence of two distinct Dirac cones ‘A’ and ‘B’ at specific k-points lying between y is invariably zero. Whereas, ky has a constant value 0.5 near the second Dirac point ‘B’. The dispersion relations between different symmetry points has been described in Table Moreover, we have plotted the dispersion relation to realize the mentclass2pt{minimF. This is a signature of particle hole symmetry in the system. Since, we have only considered the NN hoppings in a two-level system, this symmetry is well expected17. A pictorial representation of all the dispersion relations has been provided in Fig. vF) by expanding the dispersion equations in Taylor series near Dirac points. Neglecting the quadratic and higher order terms of the series we can compare the result with qx = 2πkx/a where, 2, vF = ±6 m/s 43 has previously been reported to be ≈1 × 106 It is worthy to note that the dispersion relations are symmetric about E44. During the computation, the Perdew-Burke-Ernzerhof (PBE) parameters are employed to treat the exchange-correlation part of density functional. Besides, the generalized gradient approximation (GGA) with double In order to support the band structure obtained analytically, we have performed the density functional theory (DFT) computation with the help of SIESTA packaget3 and t″ are distinct. In this case, the dispersion relation is given by Eq. t3 and t″ that allows the band gap opening in the S-graphene system. In this context, we would like to define a dimensionless parameter r that satisfies the relation Now, we want to deal with the more generic case i.e. the hopping parameters of the renormalized lattice z orbital owing to the fact that these orbitals solely contribute in the formation of Dirac cones. The NNTB Hamiltonian in the Wannier basis can thus be expressed as followsNext, we are interested to investigate the robustness of the individual Dirac cone. The underlying objective is obviously to discard the relation i-th to (i + 1)-th site. For the consistency check, we have obtained the band spectra corresponding to the Hamiltonian given in Eq. τ = 1 eV. The system exhibits the existence of two Dirac points under above conditions as depicted in Fig. τ (>t2). It has been observed that the position of the Dirac points move towards the higher symmetry points with decreasing t2. There is a critical value t2 = τ/3, however, can not split the Dirac cones for t3 < τ. This observation is also valid for t3 = 0. It can be argued that both the Dirac points are situated at the BZ path parallel to kx axis. Hence, breaking of the periodicity along the y-axis (i.e. t3 = 0) transforms the sheet into 1D nanoribbon of particular width. This ribbon will also exhibit Dirac cones. In contrary, the condition t3 > τ, induces a band gap in the system above t3 = 2τ. The above criteria is also valid for t4. In addition, we have considered the asymmetrical hopping parameters and obtained that the Dirac point A is robust to the parameter t4 upto the relation t4 = 2t′. Here, we have considered, t1 = t3 = t′ and t, t2 ≥ t′.Here, mentclasspt{minimav ≤ v < 0 protects the Dirac point B. Here, v is the ratio between the non-zero vertical renormalized hopping parameter  ≤ t and t3 ≤ 2t2 removes the degeneracy of A but not B. These results are listed in Table F giving rise to the significant change in their carrier mobility. In order to realize the feasibility of such hopping relations in band gap tuning we have applied stress in the system along x-axis using DFT. We have observed (not shown here) that 1% strain can split the Dirac cones. The result is consistent as the hopping parameter strongly depends on the separation between the neighbouring atoms.Additionally, we have explored two distinct conditions that lift the degeneracy of one Dirac point but other one remain unaltered. The analytical calculation reveals that the condition 0 < entclass1pt{minimaε is connected with the three neighbouring atoms with on-site potential −ε and vice versa. We have observed that even a small anisotropy in the on-site potential can induce band opening in the system as depicted in Fig. Subsequently, the on-site symmetry within the unit cell has been broken, while the hopping parameters remain uniform. Therefore, the atom with on-site energy +t1) only, then the system exhibits semimetallic band structure and the particle-hole symmetry is restored. This unreal particle-hole symmetry can be destroyed in presence of next nearest neighbour (NNN) hopping  of the tetragonal lattice identical to S-graphene. The exact RSRG scheme efficiently reduces the degree of difficulty of the model from eight atoms per unit cell to the two atoms per unit cell. Moreover, the NNTB Hamiltonian of such two-level tetragonal system essentially exhibits two distinct Dirac points in the (IBZ). As an important note, the Fermi velocity of the SG system (


Although there was a low effect size (2 = 0.153) was found between the crossing method and the minima and functional minima methods. This indicates that those methods assess different events during the turn switch phase. In conclusion, the sensors and assessment methodologies compared in this study are not interchangeable with the possible exception of the minima and functional minima assessed with FP.Several methods to determine turn switch points during alpine skiing using the vertical GRF exist in the literature. Although comparative studies between pressure insoles (PI) and force platforms (FP) have been conducted, there are no reports comparing the detected time points. Yet, these sensors and methods have been used interchangeably. This study aims to compare the turn switch time points with both sensors and various methods. Twenty skiers performed turns with FP and PI for two different ski styles (high and low dynamic turns). Three different assessment methodologies were compared: minima, functional minima, and crossings. Bland Altman and repeated measures ANOVA were used to assess statistical differences. Main effects of sensor and method were observed ( In sports, the determination of performance usually depends on small details. Consequently, qualitative assessment of the factors influencing performance is necessary for both recreational and elite levels. The characteristics of alpine skiing make it challenging to study the different features related to performance, injuries, or coaching. Over the last decades there have been several studies using sensors that could collect data while skiing .A total of 20 skiers  took part in the study. Before the measurement, all participants were informed in detail about the testing procedures, as well as possible benefits and risks of the investigation prior to signing the consent form approved by the local Ethics Committee. The experiment was conducted in accordance with the Declaration of Helsinki.The measurements were performed for 10 days within 2 weeks. The snow temperature was consistent across all measurement days with a mean temperature of −1.1 ± 2.2°C. The mean air temperature was −0.4 ± 4.8°C. The upper part of the measurement slope had an inclination of 23° while the bottom part had an inclination of 15°. The slope was machine groomed daily resulting in a compact layer of natural and artificial snow.Two different skiing styles were performed. The skiing technique “Carving in Short Radii”  used recreational skis  and the skiers classified as experts (n = 10) used racing skis . Regardless of the assessed level, all skiers were able to properly perform the required ski styles. The skiers used their own ski boots. The skis were equipped with a portable FP system and a pair of PI were placed inside the liners  mounted under the toe and heel binding of each ski. Each dynamometer weighed 0.9 kg, was 36 mm high, and consisted of a top and bottom plate connected by three three-dimensional piezoelectric force transducers. Amplifiers, power supply, supply box, and data loggers (4 kg) were carried in a backpack worn by the skiers . Prior to the application of the turn detection methodologies, the data sets where synchronized (based on GRF peaks produced by the stomps). The force values of the PI are the sum of the force from each cell, which is obtained multiplying its pressure by its area.The three different turn switch methods assessed were :GRF minima. This method uses the minimum value of the vertical component of the GRF determined by the sum of forces from both legs , low dynamic turns , and for all turns pooled together .Data processing was performed using IkeMaster  and Matlab . To assess the interchangeability and agreement between methodologies and sensors, the bias, and limits of agreement were calculated as proposed by Bland and Altman  . This reference was calculated independently for each turn and cut-off frequency as the mean of the 6 measured turn switch points  and partial eta squared   was used to assess statistical differences with respect to system (FP vs. PI), calculation method , filter (1 vs. 3 vs. 6 Hz), and ski dynamics (high vs. low dynamic turns). Mauchly's Test of Sphericity indicated that the assumption of sphericity was not met, consequently Greenhouse-Geisser correction was utilized. If significant differences were found, Bonferroni's The agreement between sensors and methodologies are presented in The comparison between FP and PI for all turns pooled together yielded bias lower than 0.05 s and ranges between 1.09 and 1.36 s, except for the “crossing” method with ranges starting at 0.58 s. The “minima” and “functional minima” methods had the highest agreement for both sensor types (FP and PI). Although the comparison between “minima” and “functional minima” methods had the highest agreement for both sensors, the results were not similar. Including all cut-off frequencies, the bias and range were <0.002 and 0.400 s for the FP, but >0.012 and 0.850 s for the PI, respectively. This behavior was consistent, the comparison between assessment methodologies showed systematically lower bias and range values when the FP system was used.p < 0.001), but the effect size was small (p < 0.001) with a large effect size . On the other hand, the differences between both the minima and the functional minima and the crossing method were >0.13 s with a large effect size (η2 = 0.153).Results for the mean and standard deviation for each assessment option are shown in The average turn duration for high and low dynamic turns was 1.66 ± 0.27 and 2.55 ± 0.32 s, respectively. The range between FP and PI for the most similar methodologies  represents ~30 and 40% of turn duration for high and low dynamic turns, respectively. The range between minima and functional minima assessed using the PI represents a 33 and 48% of the turn duration for high and low dynamic turns, respectively, and 13% of the turn duration when assessed with the FP, independent of the turn dynamics.The current study focused on the comparison between time points of the turn switch during alpine skiing assessed with different methodologies and using FP and PI. The results showed that all the methods yielded better agreement when the data was collected using the FP than when it was collected using the PI . This trn = 2,472) lead to a type 1 error, were negligible differences are deemed statistically significant. Regardless of the risk of type 1 error, and to the potential misinterpretation of the data, the magnitude of the ranges yielded by the Bland and Altman comparisons show a CI that represents between 30 and 40% of the total turn duration. Consequently, due to the lack of agreement between sensors, these sensors should not be interchanged when comparing turn switch points or metrics measured turn by turn as the cutting point will affect the calculations . A possible interpretation of those results is that the large number of turns included in the analysis (2 > 0.153) and not between the minima and functional minima . Nevertheless, the ranges between minima and functional minima assessed with PI represents >30% of the turn duration which leads to the conclusion that they are not interchangeable. On the other hand, when using the FP, the range represents a 13% of the turn duration, which depending on the aim of the turn detection, could allow for the use of both the minima and functional minima methods interchangeably from FP measurements.The same discussion is relevant for the comparison between the methods. Although there is a main effect of methodology, with a large effect size (0.427), the differences are mostly with respect to the crossings method  highlight the differences of the points actually detected. Unfortunately, we cannot determine which sensor or method would be most representative of the true turn switch point due to the lack of valid reference data, both in the literature and this study. Based on the results, the methodology based on the minima seems to be the most appropriate to apply in the field. It presents the lowest bias values between the two sensors and also the smallest range for highly dynamic turns. On the other hand, both sensors present some advantages and disadvantages. FP showed consistently better results, yet the use of FP in training is not a feasible option. It changes the height of the bindings, adds weight to the skis and implies more weight and wires in the skier's body. For this reason, the implementation of a less cumbersome pressure sensor outside of the boot could potentially solve the problem of the missing forces through the boot cuff and provide reliable data similar to the FP. A future study using a gold standard methodology for the location of the turn switch point needs to be defined and consequently compared with the various methodologies applied for detecting the turn switch.The aim of the study was to assess if sensors (FP and PI) or assessment methods  could be used interchangeably, and according to the results of this study, they are not. The only exception being the minima and functional minima methods assessed with FP. The results of the study also suggest that the use of the FP sensor to determine turn switches based on GRF is recommended. Further research is needed to evaluate the precision of the different systems and determine which assessment method is most correlated with the real turn switch point.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Ethics Committee of the University of Salzburg. The patients/participants provided their written informed consent to participate in this study.AM and TS: conceptualization and formal analysis. AM, KN, PS, and TS: methodology. AM: software and visualization. AM, CS, and TS: investigation. TS: resources, supervision, and funding acquisition. PS: data curation. AM: writing—original draft preparation. TS, CS, PS, and KN: writing—review and editing.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.


Pleurotus ostreatus is presented as a case study, and its potential role in regulating the ligninolytic system, secondary metabolism, development, and fruiting body initiation are discussed. We propose that deciphering the nature of effector-like SSPs will contribute to our understanding of development and communication in saprophytic fungi, as well as help, to elucidate the origin, regulation, and mechanisms of fungal-host, fungal-fungal, and fungal-bacterial interactions.Small secreted proteins (SSPs) comprise 40–60% of the total fungal secretome and are present in fungi of all phylogenetic groups, representing the entire spectrum of lifestyles. They are characteristically shorter than 300 amino acids in length and have a signal peptide. The majority of SSPs are coded by orphan genes, which lack known domains or similarities to known protein sequences. Effectors are a group of SSPs that have been investigated extensively in fungi that interact with living hosts, either pathogens or mutualistic systems. They are involved in suppressing the host defense response and altering its physiology. Here, we aim to delineate some of the potential roles of SSPs in saprotrophic fungi, that have been bioinformatically predicted as effectors, and termed in this mini-review as “effector-like” proteins. The effector-like Ssp1 from the white-rot fungus  The success of fungi to adapt and proliferate in diverse ecological niches is dependent on their ability to respond to changes in the environment. The rich, yet versatile, fungal secretome is an important component that facilitates the fast adjustment to changes occurring in the vicinity of the growing and developing fungus . The sizSSPs are secreted by fungi independently of their lifestyle and are defined as proteins that contain a signal peptide and a sequence of less than 300 amino acids. Many SSPs are coded by orphan genes, lacking known PFAM domains or similarities to known sequences. The proportion of SSPs ranges from 40 to 60% of the secretome across all lifestyles and phylogenetic groups within the fungal kingdom . SSPs haHere, we follow the common paradigm defining effectors as SSPs used by a range of pathogenic and beneficial fungi to alter the physiological status of the host plant . We specSSPs functioning as effectors are common in several plant pathogens . Many efDuddingtonia flagrans, 117 effectors were predicted, of which PefB was induced during interaction with the host and was imported into host nuclei in vitro  . The besoot tips . It is snization . MiSSP8 ormation .Rhizophagus irregularis SP7 protein, manipulates the ethylene-signaling pathway and is localized to the host nucleus where it subsequently reshuffles plant defense pathways  or by overexpression of the dominant Ssp1 member (OEssp1)  . Analysitabolism , along wtabolism . Overexptabolism , which itabolism . Howevertabolism .SSPs are a significant, yet still a functionally-elusive, part of the fungal secretome, especially in saprophytes. Nonetheless, some of them are the likely ancestors of the better understood effectors in pathogenic and mutualistic fungi . Part ofP. ostreatus highly resemble the mentioned characteristics of QS , but can be carried out in a growing array of SSP-producing fungi, such an Armillaria ostoyae, Schizophyllum commune, and more , imposing technical difficulties when it comes to their genetic manipulation. In order to uncover and validate their role(s) and function(s), the research must be accompanied by a “wet bench” experimental approach. Perhaps some of these can be achieved by using RNAi and/or CRISPR-Cas9 technologies  interactions.DF, OY, and YH wrote this mini-review together.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.


Studies on the prognostic value of the soluble programmed death ligand 1 (sPD-L1) in cancer patients have not yielded consistent results.This meta-analysis was performed to assess the association between sPD-L1 and the prognosis of cancer patients.Published articles in Pubmed, EMBASE, and Cochrane clinical trial databases were searched from the inception to September 2020. Overall survival (OS), progression-free survival (PFS), recurrence-free survival (RFS), and disease-free survival (DFS) data were evaluated using a hazard ratio (HR) at 95% confidence interval (95% CI).2 = 33%). High sPD-L1 had worse PFS , and worse DFS , without significant statistical difference in RFS .A total 31 studies involving 17 tumors and 3,780 patients were included. The overexpression of sPD-L1 was associated with shorter OS (HR 1.85, 95% CI 1.59–2.15, IHigh sPD-L1 levels were associated with worse survival prognosis in cancer patients. The sPD-L1 may be a potential prognostic, non-invasive, and dynamic monitoring biomarker for cancers in the future. Immune checkpoint inhibitors such as anti-PD-1/PD-L1 have remarkable clinical benefits in a variety of tumors . The expPD-L1 exists in two forms; membrane-bound and soluble form . StudiesStudies on the relationship between sPD-L1 and the prognosis of cancer patients have yielded contrasting results. In NSCLC, studies by He et al. showed that patients with high sPD-L1 have a reduced risk of death . HoweverRelevant articles published in various databases were searched since the inception of the databases to September 2020. The Pubmed, EMBASE, and Cochrane clinical trial databases were searched for the meta-analysis. The Clinicaltrial.gov, American Society of Clinical Oncology (ASCO) and ESMO databases were also searched. Search terms were composed of various combinations of “soluble PD-L1”, “sPD-L1”, “serum PD-L1”, “plasma PD-L1”, “circulatory PD-L1”, “blood PD-L1”, “programmed death ligand 1”, “B7-H1” and “cancer”, “neoplasm”, “carcinoma”, “lymphoma”, “sarcomas”. The language of the article was not restricted.The inclusion criteria for various studies were: i. The included patients were all pathologically diagnosed tumor patients; ii. With analyzable soluble PD-L1 data; iii. With sufficient clinical features and data that could be combined to analyze soluble PD- L1 and survival prognosis; iv. Had their sPD-L1 levels detected by enzyme-linked immunosorbent assay (ELISA). The exclusion criteria were: i. Case reports, reviews, and tissue immunohistochemical (IHC) detection for PD-L1; ii. Animal experiments; iii. No direct analysis of the relationship between soluble PD-L1 and survival prognosis; iv. Incomplete data or non-original research.First, the outcomes of each study were extracted, including overall survival (OS), progression-free survival (PFS), recurrence-free survival (RFS), and disease-free survival (DFS) prognosis data. Next, the baseline data of the studies were extracted and analyzed; publication years, study type, patient characteristics, treatment methods, and the cut-off value of sPD-L1, etc. Based on the purpose of our research, OS was the primary outcome of interest. All data screening and extraction processes were independently performed by two investigators.The Newcastle-Ottawa Quality Assessment Scale (NOS) was used to assess the quality of the study. The NOS consists of three parts: selection (4 points), comparability (2 points), and outcome evaluation (3 points). 2 statistics. I2 >75% indicated considerable heterogeneity  of all results was calculated. All outcomes were directly extracted from the studies. Time-to-event endpoints  were pooled with the use of hazard ratio (HR). Heterogeneity was assessed using the χ2-based Q test and Iogeneity . The ranWe initially identified 926 articles from the searched databases, and after excluding duplicates, 642 articles remained. After excluding 514 records, the full text of 128 articles were screened. After full-text screening, 97 studies were excluded due to the following reasons; not original studies n = 11), irrelevant studies n = 24), irrelevant outcomes n = 32), incomplete data n = 17), and the cohort overlaps n = 13). Finally, 31 studies involving 17 tumors and a total of 3,780 patients met our inclusion criteria Figure 1, irrelev. Finally, and the, irrelev, incomplThe main characteristics of the 31 studies are summarized in 2 = 33%)  Figure 22 = 83%). Besides, six studies included DFS data. High sPD-L1 was correlated with worse DFS . Moreover, only two studies reported RFS, and sPDL1 was insignificantly correlated with a worse RFS   Table 2.2 = 21%) and prospective studies  was correlated with worse OS. Studies were stratified according to whether surgery was performed, and the results showed that surgery did not affect the correlation between sPD-L1 and OS   Table 3.2 = 45%) and multivariate analysis  . We also performed subgroup analyses based on cut-off points . We found that the significant association between sPD-L1 and survival did not change with the change in cutoff points. Furthermore, sPD-L1 was associated with worse OS in both univariate  Table 3.This meta-analysis summarizes the correlation between the level of sPD-L1 and the prognosis of various cancer patients. The main significance of sPD-L1 detection is to establish a minimally invasive, easy-to-clinical, and predictive immune biomarker for cancer patients.PD-L1 can be divided into membrane-bound PD-L1 (mPD-L1) and sPD-L1. It is believed that sPD-L1 is mainly produced by the proteolysis of mPD-L1. The immunosuppressive correlation and prognostic values of mPD-L1/sPD-L1 have not been established. Currently, IHC detection is commonly used in clinical practice to detect mPD-L1. Previous studies have explored the relationships between mPD-L1 expression levels in tumor tissues and clinical prognosis. However, these studies are limited by difficulties in sampling tumor tissues, as well as by the inability to dynamically detect changes in patients during treatment. In addition, the relationships between the levels of PD-L1 as detected by tissue IHC and the prognosis of cancer patients are not clinically binding , 43. UncWe found that high sPD-L1 levels can be used as a prognostic biomarkers for poor treatment outcomes. Secondly, it has been proved that the sPD-L1 still retains its biological activity. We can further postulate that sPD-L1 in peripheral blood can be able to specifically bind T cells in peripheral blood, thereby, inhibiting T cell activity and inducing systemic immune suppression . When PDSubgroup analyses were performed based on the treatment or cut-off value of the patient. In the subgroup analysis of patients receiving immunotherapy, patients with high sPD-L1 levels were correlated with a worse OS. An NSCLC study showed that the death risk in patients with high levels of sPD-L1 was 2.68 (95%CI 1.36–5.28) times the risk in patients with low sPD-L1 levels . Given tsPD-L1 can be used as a good indicator for the survival of cancer patients. Several studies have evaluated the predictive role of other immune-related biomarkers  in different types of cancers . Meyo etTwo systematic reviews and meta-analysis have evaluated the relationship between sPD-L1 and tumor prognosis. The review by Ding et al. in 2017 included eight articles with a total of 1,102 cancer patients . Wei et This study has certain limitations. First, the studies included in this meta-analysis were observational studies, due to the lack of relevant randomized controlled trials (RCTs). Higher quality RCTs are needed to further evaluate the prognostic value of sPD-L1. Second, the number of patients with each tumor included in this study was small, and a larger sample population is needed for verification. Third, different tumor types have different molecular signatures including immune checkpoint regulators, therefore, combining them leads to inaccurate inferences and misleading clinical applications. Finally, the cut-off values of sPD-L1 in different studies were significantly different, leading to limitations in clinical applications. In future, more studies should aim at accurately establishing the correlation between sPD-L1 expression level and prognosis. However, this study shows the prognostic value of sPD-L1 in a variety of tumors and immunotherapy, indicating that sPD-L1 can potentially serve as an innovative biomarker for predicting the prognosis of cancer patients.In conclusion, this meta-analysis revealed that high sPD-L1 levels were associated with worse survival outcomes  in cancer patients. And among patients who had received immunotherapy, patients with high sPD-L1 levels had worse OS. The sPD-L1 may be a potential prognostic, non-invasive, and dynamic monitoring biomarker for cancers.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.PH, HL: protocol/project development. PH, HL, YW: data collection and management. HL, WH: data analysis. HL, WH, YZ: manuscript writing/editing. All authors contributed to the article and approved the submitted version.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.