Arabidopsis immune receptors RRS1 and RPS4 detect the Pseudomonas syringae pv. pisi secreted effector AvrRps4 via a WRKY ID in RRS1. We used structural biology to reveal the mechanisms of AvrRps4C–WRKY interaction and demonstrated that this binding is essential for effector recognition in planta. Our analysis revealed features of the WRKY ID that mediate perception of structurally distinct effectors from different bacterial pathogens. These insights could enable engineering NLRs with novel recognition specificities, and enhance our understanding of how effectors interact with host proteins to promote virulence.This study reveals a mechanism for effector perception by a plant NLR immune receptor that contains an integrated domain (ID) that mimics an authentic effector target. The  Arabidopsis NLR pair RRS1-R/RPS4 confers disease resistance to different bacterial pathogens by perceiving the structurally distinct effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia solanacearum via an integrated WRKY domain in RRS1-R. How the WRKY domain of RRS1 (RRS1WRKY) perceives distinct classes of effector to initiate an immune response is unknown. Here, we report the crystal structure of the in planta processed C-terminal domain of AvrRps4 (AvrRps4C) in complex with RRS1WRKY. Perception of AvrRps4C by RRS1WRKY is mediated by the β2-β3 segment of RRS1WRKY that binds an electronegative patch on the surface of AvrRps4C. Structure-based mutations that disrupt AvrRps4C–RRS1WRKY interactions in vitro compromise RRS1/RPS4-dependent immune responses. We also show that AvrRps4C can associate with the WRKY domain of the related but distinct RRS1B/RPS4B NLR pair, and the DNA-binding domain of AtWRKY41, with similar binding affinities and how effector binding interferes with WRKY–W-box DNA interactions. This work demonstrates how integrated domains in plant NLRs can directly bind structurally distinct effectors to initiate immunity.Plants use intracellular nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)–containing immune receptors (NLRs) to detect pathogen-derived effector proteins. The  Plants coevolve with their pathogens, resulting in extensive genetic variation in host immune receptor and pathogen virulence factor (effector) repertoires . To enabNLRs typically contain an N-terminal Toll/interleukin-1 receptor (TIR) or coiled-coil (CC) domain, a central NBD , and a C-terminal LRR domain . In addi10Arabidopsis NLR pair RRS1-R/RPS4 is a particularly interesting NLR-ID/NLR pair that confers resistance to bacterial pathogens Pseudomonas syringae and Ralstonia solanacearum, and also to a fungal pathogen (Colletotrichum higginsianum) where the effector is unknown (WRKY), which interacts with two structurally distinct type III secreted bacterial effectors, AvrRps4 from P. syringae pv. pisi and PopP2 from R. solanacearum (WRKY domain may mimic the DNA-binding domain of WRKY transcription factors (TFs), the putative virulence targets of AvrRps4 and PopP2, to enable immune perception of these effectors . RRS1-R, from the accession Ws-2, has a 101–amino acid C-terminal extension beyond the end of the WRKY domain, and can perceive AvrRps4 and PopP2, while RRS1-S from Col-0, which perceives AvrRps4 but not PopP2, is likely a derived allele with a premature stop codon, and has only an 18–amino acid C-terminal extension  and an 88–amino acid C-terminal fragment (AvrRps4C)  , 26. Preesponses , 26. AvrAvrRps4C , 28. Poptermined , but howC recognition by the integrated WRKY ID of RRS1. The recognition of AvrRps4C is mediated by the β2-β3 segment of RRS1WRKY, the same region used to bind PopP2. This segment interacts with surface-exposed acidic residues of AvrRps4C. Structure-informed mutagenesis at the AvrRps4C–RRS1WRKY interface identifies AvrRps4 residues required for protein–protein interactions in vitro and in planta and AvrRps4 perception and immune responses. Residues mediating the interaction of AvrRps4C and RRS1WRKY are conserved in both the RRS1BWRKY and the DNA-binding domain of WRKY TFs, and AvrRps4C mutants that prevent interaction with RRS1WRKY also disrupt binding to AtWRKY41. This supports the hypothesis that the RRS1WRKY mimics host WRKY TFs through a shared effector-binding mechanism. We also show that AvrRps4C prevents the interaction of RRS1WRKY and AtWRKY41 with W-box DNA, most likely via steric blocking, at the same WRKY domain site acetylated by PopP2.Here, we determined the structural basis of AvrRps4C interacts with the RRS1WRKY domain, constructs comprising residues 134 to 221 of AvrRps4C  and residues 1194 to 1273 of RRS1-R (corresponding to the RRS1WRKY domain) were separately expressed in Escherichia coli and proteins were purified via a combination of immobilized metal-affinity chromatography (IMAC) via 6×His tags and gel filtration (Superdex 75 26/60 and Superdex S75 16/60) . We qualitatively assessed the interaction of purified AvrRps4C with RRS1WRKY using analytical gel filtration chromatography. Individually, the proteins displayed well-separated elution profiles. RRS1WRKY eluted at a volume (Ve) of 14.9 mL and AvrRps4C eluted at a Ve of 12.1 mL  of 0.103 μM .We then determined the binding affinities of the interaction using isothermal titration calorimetry (ITC). Titration of AvrRps40.103 μM B and stopitation , 30]. WeC and RRS1WRKY interaction, we coexpressed the proteins in E. coli, purified the complex, and obtained crystals that diffracted with 2.65-Å resolution at the Diamond Light Source . The crystal structure of the AvrRps4C–RRS1WRKY complex was solved by molecular replacement using the structure of RRS1WRKY  and AvrRps4C (PDB ID code 4B6X) as models . X-ray data collection, refinement, and validation statistics are shown in SI Appendix, Table S2.To reveal the molecular basis of the AvrRps4C and RRS1WRKY . Also, RRS1WRKY adopts a conventional WRKY domain fold  and comode 2AYD ] compris complex   and 11.9% (593.9 Å2) of the total accessible surface areas of the effector and integrated domain, respectively [as calculated by PDBePISA . The interaction between the β2-segment of RRS1WRKY, which harbors the WRKYGQK motif, and AvrRps4C includes hydrogen bonds and/or salt-bridge interactions involving Tyr1218 and Lys1221 of RRS1WRKY and AvrRps4 Glu175, Glu187, and Asn171. Notably, the side chain of RRS1WRKY Lys1221 protrudes into an acidic cleft on the surface of AvrRps4C to contact the side chains of both AvrRps4 Glu175 and Glu187 (Movie S1). The OH atom of RRS1WRKY Tyr1218 forms a hydrogen bond with the ND2 atom of AvrRps4 Asn171  and tested the effect on protein interactions by ITC. Each AvrRps4C mutant was purified from E. coli under the same conditions as for the wild-type protein, and proper folding was evaluated by circular dichroism (CD) spectroscopy . ITC titrations were carried out as for the wild-type interactions. Individual ITC isotherms are shown in SI Appendix, Table S1. We found that mutating AvrRps4 residues Asp164 (D164A), Glu175 (E175A), Glu187 (E187A), and double mutant Glu175/Glu187 (EE/AA) essentially abolished complex formation in vitro  [previously identified to be essential for the virulence activity and perception of AvrRps4 , we tested the impact of the AvrRps4C EE/AA double mutant on the binding to AtWRKY41. We found that this mutant also abolishes interaction with AtWRKY41, suggesting the same AvrRps4-binding interface is shared with different WRKY proteins .Since AvrRps4C–RRS1WRKY interface observed in the crystal structure, we tested the effect of the AvrRps4C mutants above on RRS1-R/RPS4–mediated immunity by monitoring the cell-death response in N. tabacum. Agrobacterium-mediated transient expression of wild-type AvrRps4 triggers a hypersensitive cell-death response (HR) 5 d post infiltration when coexpressed with RRS1-R/RPS4 . We found that AvrRps4 variants elicited similar immune responses when transiently coexpressed with RRS1-S/RPS4 or RRS1-R/RPS4.To validate the biological relevance of the AvrRps41-R/RPS4 A. The pr1-R/RPS4 , 26 was in vitro A. Intereblotting B. In addWRKY in vivo as well as in vitro, we performed coimmunoprecipitation assays using full-length C-terminal 4×myc-tagged AvrRps4 constructs and C-terminal 6×His/3×FLAG-tagged constructs of RRS1-RWRKY+83  system (Arabidopsis ecotype Ws-2 (encoding RRS1-R/RPS4 and RPS4B/RRS1B) and Ws-2 rrs1-1/rps4-21/rps4b-1 (RRS1-R/RPS4/RPS4B triple-knockout) lines and were scored at 20 h post infiltration. Pf0 carrying wild-type AvrRps4 triggered HR in Ws-2, but not in Ws-2 rrs1-1/rps4-21/rps4b-1, as previously reported  system . HR assareported , 26. Avrreported A. None o/rps4b-1 A.Arabidopsis ecotype Col-0  and the Col-0 rrs1-3/rrs1b-1 (RRS1-S/RRS1B double-knockout) line. Overall, we observed a weaker HR toward AvrRps4 wild type and mutants in Col-0 in comparison with Ws-2. Nevertheless, a similar pattern of HR phenotypes was observed in Col-0 compared with Ws-2, and none of the AvrRps4 variants triggered HR in the Col-0 rrs1-3/rrs1b-1 line . The pattern of HR phenotypes conferred by the AvrRps4 interface mutants further validates the AvrRps4C–RRS1WRKY structure and the role of these residues in recognition of AvrRps4 by the RRS1/RPS4 receptor pair.In addition to Ws-2, we also performed a parallel set of experiments in Arabidopsis, we next investigated their effects on bacterial growth. We performed bacterial growth assays on Arabidopsis ecotypes Ws-2, Col-0, Ws-2 rrs1-1/rps4-21/rps4b-1, and Col-0 rrs1-3/rrs1b-1 using the P. syringae pv. tomato (Pto) DC3000 strain carrying AvrRps4 wild type or structure-based mutants. Since both the single mutants AvrRps4 E175A and E187A displayed the same impaired HR as the double AvrRps4 EE/AA mutant in previous assays, we focused on AvrRps4 EE/AA only for this assay. Bacterial growth was scored at 3 d post infection. Pto DC3000 carrying wild-type AvrRps4 displayed reduced growth on Ws-2 when compared with the mutant background (Ws-2 rrs1-1/rps4-21/rps4b-1), presumably due to the activation of RRS1-R/RPS4–dependent immunity  when compared with wild-type AvrRps4, even though they displayed a similar cell-death phenotype in N. tabacum  and HR in Arabidopsis .Having demonstrated the role of AvrRps4 interface residues in effector-triggered HR in immunity B. The efimmunity B. Pto DCbidopsis A and 5A.ion line B. FinallArabidopsis . To explore AvrRps4 recognition by RRS1B/RPS4B, we performed ITC titrations of RRS1BWRKY with wild-type AvrRps4C in vitro. We found that RRS1BWRKY binds to AvrRps4C three times more weakly than RRS1WRKY , possibly due to subtle changes imposed by residues outside the direct binding interface. When comparing the binding kinetics with the strength of immune responses in planta, we observed a weaker RRS1B/RPS4B-dependent HR to AvrRps4 compared with RRS1/RPS4. Nonetheless, both NLR pairs displayed a similar profile of immune responses toward the AvrRps4 structure-guided mutants in transient cell-death assays and in Arabidopsis HR assays .In addition to RRS1/RPS4, the RRS1B/RPS4B pair can confer recognition of AvrRps4 in bidopsis . SequencSI Appendix, Fig. S4 and Movie S2) and are indispensable for DNA binding  and studied their effect on the DNA-binding capacity of RRS1WRKY and AtWRKY41 using both electrophoretic mobility-shift assay (EMSA)– and surface plasmon resonance (SPR)–based assays. We found that the interaction of RRS1WRKY and AtWRKY41 with W-box DNA was reduced after preincubation with increasing concentrations of AvrRps4C but not the AvrRps4C EE/AA mutant , revealing that AvrRps4C interferes with WRKY binding to W-box DNA.To regulate gene expression, WRKY TFs bind to specific W-box DNA motifs in the promoters of their target genes –36. Intr binding . To testArabidopsis NLR RRS1 binds to the Pseudomonas effector AvrRps4, and how this underpins RRS1/RPS4-dependent immunity in planta. Further, through this work, we gained insights into interfaces in the RRS1WRKY domain that are crucial for perception of two structurally unrelated effectors from distinct bacterial pathogens, which may have implications for NLR integrated domain engineering.Despite recent advances, structural knowledge of how diverse integrated domains in plant NLRs perceive pathogen effectors is limited. Here, we investigated how the integrated WRKY domain of the C–RRS1WRKY complex reveals that the effector directly interacts with the DNA-binding WRKYGQK motif, likely rendering it unavailable for binding to DNA . AvrRps4C binds to AtWRKY41 with similar thermodynamic parameters to RRS1WRKY, and interface mutants that prevent AvrRps4C interaction with RRS1WRKY prevent interaction with AtWRKY41, supporting the hypothesis that AvrRps4C binds different WRKY proteins via a similar interface. WRKY TFs bind W-box DNA sequences in the promoters of their target genes. We used EMSAs and SPR assays to observe how the interaction of AvrRps4C with RRS1WRKY or AtWRKY41 affects the binding of these proteins to a generic W-box DNA sequence. Preincubation of RRS1WRKY and AtWRKY41 with increasing amounts of AvrRps4C reduced DNA-binding activity, whereas preincubation with AvrRps4 EE/AA showed no significant difference . WRKY domain residues interacting with AvrRps4C are well-conserved in these TFs , suggesting that AvrRps4 could interfere with and sterically block DNA binding of multiple WRKY TFs, thus promoting virulence. In addition to WRKY TFs, a recent publication suggests AvrRps4 can interact with BTS (nucleus-located Fe sensor BRUTUS) domains to affect pathogen colonization . The second lysine of the WRKYGQK motif, Lys1221, is acetylated by PopP2, abolishing the affinity of the WRKY domain for W-box DNA  and in planta (by coimmunoprecipitation), and these did not give cell death in Nicotiana species when coexpressed with either RRS1-R/RPS4 or RRS1-S/RPS4. Further, they did not give HR or restrict bacterial growth in Arabidopsis Ws-2 or Col-0 ecotypes . The N171A mutant retained the same level of binding as wild type in vitro, and displayed the same in planta phenotypes, although restriction of bacterial growth in Arabidopsis was reduced compared with wild type in both Ws-2 and Col-0 ecotypes. Finally, the Q194A mutant showed a reduced binding in vitro (approximately sevenfold compared with wild type) but maintained an HR in Arabidopsis as well as displaying a restriction of bacterial growth in Arabidopsis, albeit reduced compared with wild type. Interestingly, this mutant consistently showed a qualitative reduction in the intensity of cell death in Nicotiana. Taken together, these AvrRps4 mutations validate the complex with RRS1WRKY in that they prevent interaction in vitro and in planta, but they are not sufficient to determine whether strength of binding in vitro can directly correlate with in planta phenotypes. Further studies, including additional mutants, will be required to study this in the RRS1/RPS4 system.Studies with the NLR pair Pik from rice have shown that the strength of effector binding to integrated domains in vitro can correlate with immune responses in planta –53. Of tC alone, or in complex with RRS1WRKY, the N-terminal KRVY motif is known to be required for both the virulence activity of the effector and its perception by RRS1/RPS4  has been expanded by molecular engineering . HoweverC (Gly134 to Gln221), RRS1WRKY (Ser1194 to Thr1273), RRS1BWRKY (Asn1164 to Thr1241), and AtWRKY41 (Thr125 to Ile204) were cloned in various pOPIN expression vectors using an in-fusion cloning strategy as described in SI Appendix, Materials and Methods.For in vitro studies, the gene fragments of AvrRps4N. tabacum and N. benthamiana, domesticated genomic fragments encoding RRS1-R, RRS1-S, RRS1B, RPS4, and RPS4B were cloned into binary vector pICSL86977 under a 35S (CaMV) promoter with a C-terminal 6×His/3×FLAG tag using the Golden Gate assembly method as described (WRKY+83. Full-length AvrRps4 (P. syringae pv. pisi) was PCR-amplified from published constructs  and integrated WRKY domain of RRS1 (RRS1WRKY) were expressed in E. coli SHuffle cells. The proteins were purified via IMAC followed by size-exclusion chromatography. Purified fractions were pooled and concentrated to 15 mg/mL and used for further studies. Detailed procedures are provided in SI Appendix, Materials and Methods.Plasmids expressing the in planta processed C-terminal fragment of AvrRps4 . Diffraction data were collected at the Diamond Light Source on the i03 beamline and processed in the P61/522 space group. The structure was determined by molecular replacement using the model of a monomer of AvrRps4C (PDB ID code 4B6X) and the RRS1WRKY from the PopP2–RRS1WRKY complex (PDB ID code 5W3X) as the search model. Further details are provided in SI Appendix, Materials and Methods. X-ray data collection and refinement statistics are summarized in SI Appendix, Table S2.Crystals of the AvrRps4C–RRS1WRKY complex formation in vitro was studied using analytical gel filtration chromatography and ITC. The effect of structure-guided mutations on the AvrRps4C–RRS1WRKY interaction in vitro was investigated using ITC as described in SI Appendix, Materials and Methods.AvrRps4Agrobacterium-mediated transient cell-death assays were performed in N. tabacum and coimmunoprecipitation assays were performed in N. benthamiana. Detailed information concerning plant materials, growth conditions, plasmid construction, and immunoblotting are provided in SI Appendix, Materials and Methods.P. fluorescens Pf0-EtHAn and Pto DC3000 were used for HR or in planta bacterial growth assays, respectively. The Arabidopsis accessions Ws-2 and Col-0 were used as wild type for all the assays in this study. Further details about plant materials, growth conditions, plasmid construction and mobilization, pathogen infection assays, and bacterial growth assays are provided in SI Appendix, Materials and Methods.Bacterial strains SI Appendix, Materials and Methods.Complementary single-stranded DNA fragments encoding the W-box DNA sequence (forward strand: 5′-CGCCTTTGACCAGCGC-3′) were synthesized by IDT. EMSAs were performed using a Cy3-labeled W-box DNA probe in a reaction buffer containing 10 mM Tris⋅Cl (pH 7.5), 50 mM KCl, 1 mM dithiothreitol, and 5% glycerol as described in SI Appendix, Materials and Methods.Complementary single-stranded DNA fragments encoding the W-box DNA sequence were synthesized by IDT. For SPR assays, the forward strand encoded the W-box DNA sequence (5′-CGCCTTTGACCAGCGC-3′) while the complementary reverse strand added an extra 20-bp ReDCaT sequence (5′-CCTACCCTACGTCCTCCTGC-3′) to complement the linker DNA added to the SA chip. The double-stranded DNA was then diluted to a working concentration of 1 μM. SPR measurements were performed at 25 °C using the reusable DNA capture technique (ReDCaT) as described , 60 and Supplementary FileSupplementary FileSupplementary File


In bulk, these two-dimensional (2D) sheets stack into a three-dimensional van der Waals crystal, where relatively weak alkyl–alkyl interactions constitute the interface between these sheets. With the liquid-phase exfoliation, we are able to obtain films as thin as two molecular layers. Further using a combination of liquid-phase and mechanical exfoliation, we are able to create non-covalent sheets over a large area (>100 μm2). The ultrathin porous films maintain the single crystal packing from the macrocyclic structure and are electrically conductive. We demonstrate that this new type of 2D non-covalent porous organic framework can be used as the active layer in a field effect transistor device with graphene source and drain contacts along with hexagonal boron nitride as the gate dielectric interface.Electroactive macrocycle building blocks are a promising route to new types of functional two-dimensional porous organic frameworks. Our strategy uses conjugated macrocycles that organize into two dimensional porous sheets  Ultrathin porous films held together by non-covalent van der Waals interactions was obtained by a top-down approach, which is then utilized as channel material in a two-dimensional planar field-effect transistor device through easy stamp transfer. The top-down approaches to these materials are enabled by strong covalent bonds in the two-dimensional plane and weak van der Waals interactions between them, similar to what is seen in two-dimensional materials such as graphene and TMDs.22–27 For porous ultrathin films, the electrical conductance has not been extensively investigated.2,7,20,28,29We describe a new type of two-dimensional (2D), molecularly-thin porous organic framework that is formed from macrocyclic building blocks that assemble, through non-covalent interactions, into a porous two-dimensional plane. Covalent organic frameworks (COFs) are promising in applications due to their ability to host other functional molecules in the voids.30 Using liquid-phase and mechanical exfoliation, we create porous films that are as thin as two-layers of molecules. These new results are exciting and useful because previously we were not able to obtain such high-ordered thin porous film directly from its bulk crystal and were limited to investigating the electronic properties of this hollow organic capsules in spin-coated films. These ultrathin porous films are ordered over large areas and maintain the single crystal packing from the macrocyclic building blocks. To demonstrate the utility of this new type of ultrathin material, we fabricated 2D field effect transistor (FET) devices in which graphene is the source/drain contacts, hexagonal boron nitride is the gate dielectric interface, and the exfoliated molecular sheet is the active layer. These ultrathin self-assembled materials are efficacious at transporting electrons and will find utility in gas sensing and applications similar to traditional two-dimensional materials.Here, we explore making molecularly thin layers in which conjugated macrocycles are used as building blocks and non-covalent van der Waals interactions are the adhesive that assembles these molecules into rigid, porous layers. By adjusting the relative strengths of the interactions that direct the assembly within the plane and those holding the two-dimensional layers with respect to each other, we can exfoliate these non-covalent porous frameworks using the same means employed for traditional two-dimensional van der Waals materials.1). Characterization is contained in the ESI311 has several important molecular features in its solid-state assembly. It is a rigid and shape persistent macrocycle that has an interior and an exterior  as the solvents for exfoliation. 1 in heptane and sonicate the mixture for five minutes. We drop cast the supernatant solution on silicon wafer and examine them with AFM. Remarkably, we are able to obtain non-covalent porous organic frameworks as thin as only two molecular layers  studies to characterize the crystallinity of the as-prepared non-covalent porous 2D sheets of 1 in a more traditional organic FET with Au contacts and trichloro(octadecyl)silane coated SiO2 as the gate dielectric.] The source-drain contacts were fabricated from graphene and the dielectric interface was hexagonal boron nitride (hBN). A schematic of the device is shown in 37 to first pick up hBN and then graphene to make the hBN/graphene stack. We transfer this hBN/graphene stack onto another clean silicon substrate. Then the graphene was cut using electron beam lithography and an oxygen plasma to open a 300 nm gap between graphene electrodes  polymer as the substrate, which was then used for the stamp transferring. In this manner, we were able to transfer the ultrathin sheets (∼20 nm) onto the graphene electrodes. −4 cm2 V−1 s−1 from the transfer curve. As expected, it is somewhat lower than the electron mobility estimated from the saturation regime of the traditional OFET shown in Fig. S3.†38 Despite the small size and the nanoscale thickness, the material exhibits over 3 orders of magnitude difference in current between the off and on state of the device. The threshold voltage is about 39 V, implying that the device turns on at relatively high voltage. We surmise that the contact, through the alkane sidechains is an impediment to more efficient charge transport.We next sought to determine the ability of these non-covalent porous ultrathin layers to transport charge. Because these films are van der Waals materials, we sought to make devices with van der Waals interfaces. [The ESIWe have described here a new type of porous 2D organic framework held together by non-covalent bonding and charts a clear path to creating functional non-covalent porous 2D materials with tunable properties by increasing intermolecular forces within the plane relative to those between the planes. Through a combination of liquid-phase and mechanical exfoliation of the molecular crystal we are able to make non-covalent porous 2D sheets that can be as thin as two-layer molecules over a large area. These sheets are able to transport electrons in a field effect transistor device with graphene source-drain contacts and hBN gate dielectric interfaces. Because these materials are electrically conductive and retain the single crystal structure from macrocyclic building blocks, they will find utility as reaction chambers for studying chemistry in confined, and electrified spaces, as molecular sensors, and as electrically activated sieves.There are no conflicts to declare.SC-012-D0SC05602B-s001


Inspired by information theoretic analyses of L1 speech and language, this study proposes that L1 and L2 speech exhibit distinct information encoding and transmission profiles in the temporal domain. Both the number and average duration of acoustic syllables  were automatically measured from L1 and L2 recordings of standard texts in English, French, and Spanish. Across languages, L2 acoustic syllables were greater in number (more acoustic syllables/text) and longer in duration (fewer acoustic syllables/second). While substantial syllable reduction (fewer acoustic than orthographic syllables) was evident in both L1 and L2 speech, L2 speech generally exhibited less syllable reduction, resulting in low information density (more syllables with less information/syllable). Low L2 information density compounded low L2 speech rate yielding very low L2 information transmission rate . Overall, this cross-language comparison establishes low information transmission rate as a language-general, distinguishing feature of L2 speech. This experience-dependent L1-L2 imbalance is evident in L1 versus L2 speech patterns. Just as listeners can identify individual talkers , learned from early and extended exposure, and the second language (L2), learned from later and more limited exposurers e.g.,  and langrs e.g.,  from shors e.g., , 2010 anrs e.g., . These oThe present study seeks to identify language-general acoustic properties that distinguish L1 and L2 speech and that may underlie the observed rapid identification of speech samples as either L1 or L2 speech. Furthermore, inspired by information theoretic analyses of L1 speech and language production, this study proposes that L1 and L2 speech exhibit distinct information encoding and transmission profiles in the temporal domain. Accordingly, the present study compares speech timing patterns in L1 and L2 speech in three languages: English, French, and Spanish. This is done in terms of two key information theoretic parameters: information density (number of speech units for a given meaning) and information transmission rate (information conveyed per second). Identification of a language-general, information-driven temporal parameter along which L1 and L2 speech differ would broaden our perspective on the communicative impact of speech communication between speakers from different language backgrounds to include the impact of L1 versus L2 speech mode on the dynamics of information flow via the speech channel. This study thus represents an initial step towards characterizing the language-general L1 versus L2 speech mode in quantifiable information theoretic terms that can be automatically applied to a multi-lingual multi-talker speech corpus. This empirical base can then support further speculation and theorizing regarding cognitive mechanisms that underlie L1 versus L2 speech production and their impact on communicative efficiency  under a variety of conversational conditions.Prior work on information encoding and transmission in language and speech supports two inter-connected claims: (1) variation in complexity at all levels of linguistic structure is related to variation in predictability given the context , and (2) the distribution of information across utterances tends toward uniform density of encoding  in order to optimize information transmission via the capacity-limited speech channel . This inA related line of research operationalized information density for cross-language comparisons in terms of the number of linguistic units over which a given meaning, or text, is distributed . This re2, this view predicts different L1 and L2 patterns of phonetic reduction. While phonetic reduction is evident to some extent in both L2 and L1 speech . L1 talkers therefore exhibit reduction patterns that are finely tuned to the contours of surprisal  during L1 speech production  for L2 versus L1 speech have been shown for L2 speech in various languages, including English , SpanishBoth speech rate and phonetic reduction influence the flow of linguistic information as the speech signal unfolds in time. For a given utterance a slower speech rate necessarily implies a slower information transmission rate (fewer bits of information conveyed per unit of time). Similarly, since less phonetic reduction implies production of more phonetic material and since all phonetic material has some acoustic duration, the tendency of L2 speech to involve fully articulated instead of reduced forms also effectively decreases the rate of information transmission for L2 relative to L1 speech. Thus, the characteristically slow rate of L2 speech presumably combines with the tendency of L2 speech towards fully articulated rather than phonetically reduced forms  to produce an L2 speech mode with a very low information transmission rate. Importantly, this low L2 information transmission rate results from a combination of slow speaking rate and low information density, a combination that is contrary to the efficiency-driven trade-off between speech reduction and information density. The present study explores this information theoretic perspective on L2 speech through a close examination of syllable rate and syllable information density – two global speech properties that together influence information transmission rate – in L1 and L2 speech across three languages.English, French, and Spanish are well-suited to this multi-language analysis because all three are widely spoken as both L1 and L2 and differ notably in their sound structures. In the World Atlas of Languages Structures Online WALS, , EnglishTo recap, this study aims to identify a language-general acoustic signature of the contrast between L1 and L2 modes of speech production in terms of information encoding and transmission in the temporal-domain. Through a novel application of information theoretic concepts to both L1 and L2 speech in three languages, this study represents an initial step towards a broader goal of gaining new insights into underlying mechanisms and communicative consequences of L1 versus L2 speech production. With the empirical contours of information transmission via the L2 speech channel in sharper focus, we will be poised for further speculation and hypothesizing for this broader research agenda.https://speechbox.linguistics.northwestern.edu/#!/?goto=allsstar) (http://rpd.chass.utoronto.ca/). The complete dataset and analysis scripts (R and Praat) are freely available from the Open Science Foundation (OSF) web-based repository via the following link: https://osf.io/vawdb/.The dataset for the present study consisted of 351 observations of 21 variables. Each observation is based on a digital speech recording of a given text by a given talker, and the variables are derived from automatic phonetic measurements applied to each recording. The digital speech recordings were taken from two separate speech corpora, the Northwestern University ALLSSTAR Corpus (llsstar)  and the An overview of the 351 digital speech recordings included in this dataset is shown in http://rpd.chass.utoronto.ca/, Recordings from 103 talkers (61 recordings in French + 42 recordings in Spanish) came from the Romance Phonetics Database  and 19 L1 Spanish  talkers had average ages of 31 and 29 years, respectively. The 47 L2 French  and 23 L2 Spanish  talkers had average ages of 27 and 31 years, respectively. The L2 French talkers came from 7 different L1 backgrounds distributed as follows: Albanian (n = 1), Arabic (n = 1), Mainland Mandarin (n = 3), Czech (n = 5), English (n = 31), Russian (n = 3), and Spanish (n = 3). Of the L2 Spanish talkers, 22 had English as their L1 and one L2 Spanish talker spoke Tagalog as their L1. L2 proficiency levels are available for most of the talkers from the RPD (63/70 or 90%) showing a concentration at the intermediate and advanced/near-native levels .3 that detects intensity peaks surrounded by intensity dips of at least 2 dB and that rise above a threshold that is determined as 25 dB below the 99th quantile of the intensity maximum for the entire sound file. The 2 dB and 25 dB settings are adjustable defaults designed to minimize the influence of non-speech sound bursts in the intensity peak picking process. Furthermore, peaks that occur during unvoiced portions of the signal are excluded. The output of the Praat script provides the basis for calculating three measures of temporal structure: nsyll (number of acoustic syllables), SR (speech rate) and AR (articulation rate). SR is the rate of syllable production over the entire recording including pauses and other major disfluencies , while AR is the rate of syllable production with silent pauses and other major disfluencies removed . By definition, for any given recording, AR is greater than SR. Because pausing and other disfluencies may be more prevalent in L2 speech than in L1 speech , information rate (IR), and syllable reduction (LOSS). While articulation rate expresses the number of speech units (acoustic syllables) per unit of time, information density expresses the amount of information encoded in each speech unit. The relation between units of information  and speech units is highly complex, abstract, and involves a degree of non-compositionality that precludes a straightforward mapping. However, following an approach developed by researchers at the Laboratoire Dynamique Du Langage (CNRS / Université Lyon 2) , we can KT is the number of acoustic syllables in the current talker’s, T’s, production of text K. The interpretation of this value is the average proportion of information encoded per acoustic syllable. Relatively low information density (ID) values indicate that the current talker distributes the information contained in the text over a relatively large number of acoustic syllables each of which encodes a relatively low proportion of the total information content of the text. Conversely, relatively high information density (ID) values indicate production of relatively few acoustic syllables each of which encodes a relatively high proportion of the total information of the text.For the purposes of the present within-language comparisons, syllable information density, ID, for each talker, T, for each text, K, was expressed as the inverse of the number of acoustic syllables produced, as shown in KT, IDKT, and avgSylDurKT are the acoustic syllable information rate, information density, and average acoustic syllable duration for the current talker’s, T’s, production of text K, respectively. The information conveyed per second, IR, is thus the information encoded per acoustic syllable divided by the seconds per acoustic syllable, to yield the information conveyed per second. The units for IR are: (info/syl) / (sec/syl) = (info/syl) * (syl/sec) = info/sec.The information rate, IR, for each recording was calculated based on the average acoustic syllable duration  and information density, ID , as in A relatively high IR, can result from either a high information density , a fast articulation rate (short durations of acoustic syllables), or a combination of high ID and fast AR. Conversely, a relatively low IR can result from either a low information density , a slow articulation rate (few syllables per second), or a combination of low ID and slow AR. As discussed in great depth in In the present study, we compare information rate (IR) of L1 and L2 speech within each text . For the L2 recordings, the IR values might be expected to reflect either a compounding of ID and AR differences (if both ID and AR are lower for L2 than L1 speech) or a counteraction of ID and AR differences (if ID is higher but AR is lower for L2 than L1 speech). IR comparisons across L1 and L2 speech are therefore interpreted as indices of syllabic information transmission efficiency taking into account both duration and number of syllables produced for a given text.LOSS, as the number of acoustic syllables for a given utterance relative to the number of orthographic  syllables that constitute the linguistic structure of the utterance as in a is the number of acoustic syllables and σo is the number of orthographic syllables.Finally, syllable loss (LOSS) is a measure of syllable level phonetic reduction as expressed through the difference between the number of acoustic and orthographic syllables. Automatic measurement of speech rate based on acoustic syllable detection is an objective, purely signal driven technique that proceeds without reference to the linguistic content of the utterance, individual talker characteristics, or other parameters related to language-specific phonotactics. Due to connected speech processes that result in extensive phonetic variation , acoustic syllables are not transparently related to the underlying abstract metrical and information bearing units of language production and perception. Specifically, the number of acoustic syllables produced for a given utterance will frequently differ from the sum of canonical, phonological syllables in the words that constitute the text of the utterance based on syllable counts in, for example, a pronunciation dictionary or as counted by L1 speakers in a metalinguistic syllable-counting task . In order to relate acoustic syllables to orthographic syllables and to understand any observed variation across L1 and L2 speech in the information transmission variables that are based on acoustic syllables , we define syllable-level phonetic reduction, σFor the English recordings in the present study, the number of orthographic words and syllables was obtained using an English syllable counting function implemented in R . This fu2.Consistent with prior comparisons of articulation rate (AR) in L1 and L2 speech, the data in In addition to articulatory slowing (fewer syllables per second), L2 speech is also characterized by a consistent increase in the number of acoustic syllables produced relative to L1 speech  using generalized linear mixed effects regression models (glmmTMB package). Separate analyses were conducted for each of the four critical dependent variables, articulation rate (AR), information density (ID), information rate (IR), and syllable reduction (LOSS). In each analysis, the effects of interest were the fixed effect of Group (L1 versus L2) and its interaction with Text . Forward contrast coding was applied to both categorical fixed factors, Group (2 levels) and Text, (4 levels). A random intercept for talker was included in the models. For all four dependent variables , gaussian, beta, and gamma distributions were compared via the Akaike information criterion (AIC) resulting in selection (minimum AIC) of gaussian distributions for AR and LOSS, and gamma distributions for ID and IR. To assess the improvement in fit of the models with the interaction term , log likelihood tests (anova) were run between the model with the interaction term and the model with only the additive term . For all four dependent variables, the model fit was significantly improved with addition of the interaction term. The model comparisons and predictors in the models with the interaction term are summarized in Pairwise comparisons for the critical effect of interest  are shown in In order to gain some insight into a possible relation between L2 proficiency and the observed patterns of variation in AR, ID, IR and syllable reduction (LOSS), a subset of the data was examined by proficiency group. However, this post-hoc and unplanned division of the binary grouping variable (L1 versus L2) into more fine-grained proficiency-based sub-groups should be considered preliminary and suggestive rather than conclusive because there was no consistent and reliable proficiency assessment across the ALLSSTAR and RPD datasets, and proficiency data were not available for all talkers. The labels “Beginner,” “Intermediate,” and “Advanced/Near-L1” were intended as approximate groupings and the available data do not support identification of clear boundaries between these proficiency labels. Moreover, as noted above, no proficiency data was available for a substantial portion of the L2 talkers (approximately 17%), and the “Beginner” subgroup was very small . Notwithstanding these limitations, a subset of the data was examined in order to see if there was any indication that the observed L1-L2 differences in AR, ID, IR, and LOSS decrease with increasing L2 proficiency.The sub-dataset for this proficiency-based examination excluded L2 talkers for whom L2 proficiency information was not available (n = 28). In addition, L2 talkers at the beginner level were excluded due to the small number of L2 talkers at that level of L2 proficiency (n = 5). Finally, to avoid analyses based on very small groups, the proficiency focused analyses were conducted on data that were aggregated across texts . The sub-dataset therefore excluded data for the ENG-DHR text in order to avoid double-counting the individual talkers in the English group. The final proficiency dataset thus included data from 80.4% of the L2 talkers and 100% of the L1 talkers in the total dataset with the distribution across proficiency levels as follows: intermediate (n = 67), advanced/near-L1 (n = 68), and L1 (n = 59).Density plots for each of the four critical variables by proficiency group are shown in 3.This study set out to identify a language-general acoustic marker of L1 versus L2 modes of speech production in terms of information theoretic parameters that have proved powerful for explaining intra-language variability in L1 speech. As such, this study represents a first step towards a broader goal of understanding information transmission via an L2 speech channel. The data showed that L1 and L2 productions of the same text within each of three typologically distinct languages  diverged along two phonetic parameters that define the temporal structure of the speech signal at the utterance level: articulation rate (the number of acoustic syllables per second) and information density (the number of acoustic syllables over which the information of the text is distributed). Specifically, in comparison to L1 speech, L2 speech was produced with a slower rate of articulation (fewer acoustic syllables per second) and a lower information density (more acoustic syllables for the given text). In combination, these two salient features of utterance-level speech timing each compounded the other such that the proportion of the total information of the text that was conveyed per second, the information transmission rate, was substantially lower for L2 than for L1 speech. Notably, both L1 and L2 speech generally involved production of substantially fewer acoustic syllables than orthographic syllables (as counted from the text scripts) indicating that the lower average information density for L2 speech is related to avoidance of phonetic reduction rather than a predisposition towards syllable insertion. As discussed in the introduction, English, French, and Spanish were well suited to this cross-language comparison due to their divergence along several relevant dimensions of sound structure including segment inventory size, consonant-to-vowel ratio, complexity of syllable structure, and several phonological processes that affect connected speech timing. The convergent pattern of lower information transmission rate for L2 than L1 speech across these languages suggests that this may be a language-general distinguishing feature of L2 versus L1 speech.This investigation was inspired by prior work supporting the claim that a significant portion of surface variation within and across languages can be attributed to a principle of efficiency of information encoding and transmission see . Within From the perspective of the message encoder , both the slower rate and the increased number of acoustic syllables in L2 speech may originate from general features of L2 speech production regardless of the particular L2 being spoken and regardless of the talker’s L1. The slower articulation rate is likely related to slower processes at multiple levels, including lexical retrieval, production planning, and speech articulation  which are less practiced and therefore more resource demanding in L2 compared to L1 speech. This possibility is consistent with the notion of phonetic reduction  as an active, and therefore resource demanding, process of information transmission optimization that talkers implement during message encoding.A second possibility is that connected speech in L1 and L2 involve similar reduction-related optimization processes, but the language models from which probabilities of occurrence are derived differ due to different contexts in which the language was learned and is used. Later onset and shorter duration are defining features of L2 versus L1 acquisition, and in contrast to the typical implicit L1 learning context, the L2 learning context often involves explicit instruction with an emphasis on written forms see . All of The complexity of the language models over which surprisal/redundancy is estimated has been highlighted in several detailed studies of redundancy-driven reduction in L1 speech e.g., . Using ltraveler” in the English NWS passage is frequently pronounced with two syllables in L1 English. However, in L2 English, this word is frequently pronounced with three syllables. The unstressed middle syllable of the orthographic form is often fully articulated in L2 speech but reduced in L1 speech. Similarly, in the French NWS passage, the word “enveloppé” may be pronounced with four full syllables in L2 French but with a reduced second syllable in L1 French, and in the Spanish NWS passage, the word “viento” may be pronounced with three syllables in L2 Spanish but with two syllables in L1 Spanish. These L1 reductions may reflect redundancy, or predictability, based on lexical frequency, morphological composition, phonotactic sequencing, and/ discourse context. In contrast, for L2 talkers, their learning context and usage-based experiences may prioritize the orthographically-driven pronunciation resulting in production of “extra” acoustic syllables, thereby lowering the L2 speech information density and transmission rate.Furthermore, L2 learning often relies heavily on the written medium which could lead to prioritization of production targets based on orthographic forms with fully specified syllables over frequently used reduced forms. For example, the word “From the perspective of the listener at the receiver’s end of the speech transmission chain, what might be the consequences for message decoding of the slower information transmission rate of L2 compared to L1 speech? Studies of L2 speech recognition by L1 listeners have demonstrated highly generalized improvement in L2 speech recognition accuracy following repeated exposure to L2 speech samples e.g., , suggestThe divergence of intelligibility  and communicative efficiency  is also illustrated by the contrast between L2 speech and “clear” speech, a speech style that talkers often adopt when they are aware of a speech communication barrier for the listener due to, for example, a hearing impairment or environmental noise  and low information density (many syllables for a given message) to yield a slow information rate  for L2 speech compared to L1 speech. A key feature of the data presented in this study is that the L1 and L2 speech recordings within each of the three languages  were readings of a given text. Thus, the within-language comparisons of L1 versus L2 speech were controlled for intended meaning, allowing us to view the rate and number of acoustic syllables produced in relation to the encoding of a fixed meaning in L1 versus in L2 speech. However, this dataset was rather limited in terms of statistical modelling of phonetic reduction as a function of surprisal, or probability, of linguistic forms in various contexts. Detailed analyses with large corpora of L2 speech would allow for direct comparison of the relationship between probability of occurrence given the context  and phonetic reduction in L2 and L1 speech. This would, in turn, allow for deeper insight into the causes and consequences of the distinct temporal modulation pattern of L2 speech across various languages and for hypothesis-driven testing of alternative explanations for the observed differences between L1 and L2 information encoding and transmission4.This study provides cross-language evidence that the characteristically slow rate of L2 speech (low number of acoustic syllables per second) combines with a tendency towards fully articulated rather than phonetically reduced forms  to produce an L2 speech style with a very low information transmission rate. This compounding of slow articulation rate with low information density in L2 speech contrasts with the efficiency-driven trade-off between speech reduction and information density that characterizes L1 speech. Future research with large-scale, multi-lingual, multi-talker corpora of L1 and L2 speech under natural, conversational conditions and in a wide range of languages are needed to gain further insight into the dynamics of communicative efficiency via L2 speech channels.


The emission modes and thresholds were strongly dependent on both the graphene doping concentration and the external temperature. Decreased plasmon coupling was the primary reason for lower QD/graphene laser emission with increasing temperature. The optimum reduced graphene oxide concentration was 0.2 wt.%. This work provides a practical approach to optimizing the threshold and stability of random laser devices, with potential applications in displays, sensors, and anti-counterfeiting labels.An improvement in random lasers based on a colloidal quantum dot (QD)/graphene-doped polymer was observed and attributed to multiple light-scattering and graphene surface plasmon resonance. The emission characteristics of quantum dots doped with graphene oxide and reduced graphene oxide were compared. The QD/reduced graphene oxide hybrid exhibited a lower laser emission threshold (~460 μJ/cm In contrast to conventional lasers that require an optical resonator with two highly reflective mirrors to confine the photons, random lasers are cavity-free and can be achieved by extended or localized modes via multi-scattering in a disordered gain medium ,2,3. RanMechanisms and structures of random lasers have been extensively studied, including Anderson light localization to glassy-light behavior and the interplay between homogeneously dispersed plasmonic nanoparticles ,10,11,122 bonding of graphene can be interrupted by chemical oxidation [2 clusters within the graphene oxide could serve as surface plasmon (SP) interaction centers and induce random laser activity [The wrinkled structures within graphene and its porous vertical network induce multi-scattering of light emitted from the gain material and confine the photons to enable stimulated emission ,21. In axidation . The embactivity ,24. Impractivity ,26. The Here, ZnCdSeS/ZnS alloy quantum dots and graphene were simultaneously dispersed in a prepolymer monomer that was subsequently polymerized. After the ultraviolet curing process, the homogeneous quantum dot/graphene-doped polymer was pumped with a nanosecond laser. The random lasing thresholds and PL intensities from the samples were measured with respect to the morphologies of graphene and its derivatives. The coupling effect of graphene-based plasmons and colloidal quantum dots was discussed. We also addressed the temperature effects on the resonance characteristics of the random lasers. The emission modes and thresholds were strongly dependent on both the graphene-doping concentration and the external temperature. This work presented a simple method for fabricating a low threshold random laser from polymer doped with quantum dots and graphene. Because of its good stability, short production period, and versatility, it could be developed into a prototype for random fiber lasers, sensors, anti-counterfeit labels, and displays.2).ZnCdSeS/ZnS quantum dots were prepared as reported previously . Figure XRD characterization was used to indirectly analyze the graphene exfoliation and dispersion in the polymer matrix. As shown in −1 corresponding to G and D bands. The G band was generated from the vibration of sp2-bonded. The D band was generally correlated with defects derived from vacancies, grain boundaries, and amorphous carbon species [D/IG ratio of the GO was lower than that of rGO, indicating that the number of smaller graphene domains increased [Raman spectra of GO and rGO were measured by a Raman spectrometer with laser of 532 nm excitation. As displayed in  species . The ID/ncreased .2. No sharp peaks were observed at low pump energies. Above the laser threshold, the QD-GO and QD-rGO samples exhibited coherent random-lasing and numerous sharp emission peaks appeared. The FWHMs of spikes were less than 0.1 nm , as shown in The effect of rGO concentration on the emission characteristics of QD-rGO samples was examined for additional rGO doping at 0.05 wt.%, 0.1 wt.%, 0.2 wt.%, 0.3 wt.%, and 0.4 wt.%, as shown in 2). The optimum doping concentration of rGO in the polymer was 0.2 wt.%. Overall, this work provides a practical approach for optimizing the threshold and stability of random laser devices, with potential applications in sensors and confidentiality tags.We demonstrated improved random laser activity using a polymer doped with colloidal quantum dots and graphene. In this system, graphene was the scattering medium and supplied SPR coupling with the lasing emission. We could conclude from the temperature-dependence experiments that reduced SP coupling was the primary cause for decreasing QD-graphene laser emission with increased temperature. The plasmon density of states and the SP-QD coupling efficiency strongly depended on the graphene’s number and types of surface functional groups; hence, QD-rGO had a lower laser emission threshold (~460 μJ/cm


Aims: Current idiopathic pulmonary fibrosis (IPF) therapies usually show a poor outcome or treatment efficacy. The search for new risk factors has significant implications in preventing, delaying, and treating IPF. The association between obesity and the risk of IPF is not clear. This study aimed to investigate the role of different obesity types in IPF risk, which provides the possibility of weight loss as a new approach for IPF prevention.Methods: We conducted a two-sample Mendelian randomization (MR) analysis to assess the causal effect of obesity on IPF risk. We collected summary data of genetically determined obesity-related traits, including body mass index (BMI), waist circumference (WC), and waist-to-hip ratio (WHR) from large-scale consortia , and genetic association with IPF from one of the largest meta-analyses including 2,668 cases. A total of 35–469 single nucleotide polymorphisms were selected as instrumental variables for obesity-related traits. We further performed multivariable MR to estimate the independent effect of BMI and WC on the risk of IPF.Results: Increased BMI and WC were associated with higher risk of IPF . Similar results for the BMI and WC were obtained in the replicated analysis. Subsequently, only the result for BMI survived following the multiple testing correction and showed good consistency with the weighted median estimator. Sensitivity analyses indicated that there was no heterogeneity or horizontal pleiotropy for MR estimations. Further multivariable MR suggested that the BMI showed the same direction and similar magnitude with that in the univariable MR analysis. There was little evidence to support the causal role of WHR on the risk of IPF in this study.Conclusion: Genetically determined BMI demonstrates a causal risk for IPF, which offers a novel insight into probing potential mechanisms. Meanwhile, these results also suggest that weight loss may be beneficial to IPF prevention. Idiopathic pulmonary fibrosis (IPF), a kind of idiopathic interstitial pneumonia with diffuse parenchymal abnormalities and lesions, is the most frequent and severe form of fibrotic lung disease . The incIn a phase III trial of pirfenidone in 1,247 patients with IPF, the proportion of patients with obesity was 44% , suggestMR is considered as “nature's randomized control trial” , using ghttp://portals.broadinstitute.org/collaboration/giant/index.php/GIANT_consortium_data_files) and the MRC Integrative Epidemiology Unit (MRC-IEU) using the United Kingdom Biobank genetic data (https://www.mrbase.org/)  was set to ensure that the genetic instruments selected for obesity are conditionally independent to each other. The F statistic represents the strength of the relationship between IVs and VAT. Generally, F > 10 may attenuate bias of using weak genetic instruments  from the Genetic Investigation of ANthropometric Traits (GIANT) consortium (se.org/) . The GIAtruments . Finallytruments , 40 SNPstruments , and 35 truments  as IVs tWe collected the summary data of IPF from the largest available GWAS to date, which was a meta-analysis of three studies including 2,668 IPF cases and 8,519 controls . Of thescausal effect = βZY/βZX . Generally, a valid IV should satisfy three assumptions (p < 5 × 10–8) (As shown in umptions : must be × 10–8) ; not ass × 10–8) ; and sho × 10–8) .To evaluate the causal effects of obesity-related traits on the risk of IPF, we conducted a two-sample MR analysis  using thMoreover, we used summary data from two additional studies to extract IVs for BMI  and WC  analysis to estimate the relatively direct causal effect of BMI and WC on the risk of IPF.p-value < 0.017 (0.05/3)  ; IVW and(0.05/3) ; the oth(0.05/3) .p-values were two-sided, and the statistical significance was set at the adjusted p-value < 0.017.MR analysis was performed in R (version 4.0.3) with R packages “vroom,” “tidyr,” “tibble,” “dplyr,” “TwoSampleMR” , “MR-PREF statistics ranging from 35.62 to 81.87, reflecting a strong instrument strength for obesity. A total of 66 SNPs for BMI and 335 SNPs were selected for WC extracted from the other independent GWAS summary data for further replicated analysis, with F statistics of 344.85 and 50.94, respectively.The characteristics of the participants from the GIANT consortia for obesity-related traits and meta-analysis for IPF are shown in p = 1.27 × 10–4] and 1.71 , respectively. However, there is little evidence to support an association between WHR (adjusted for BMI) and IPF. We obtained similar results in the replicated analysis, suggesting that the increased BMI and WC are associated with IPF risk . Of these, the MR method of weighted median showed good consistency with IVW (p < 0.05) for the BMI. Subsequently, only the results for BMI and IPF survived following the multiple testing correction .The overall results of the univariate MR analysis for genetically determined obesity on IPF risk are shown in pheterogeneity > 0.05) for the presence of heterogeneity for the MR estimations of BMI, WC, and WHR. Additionally, the MR-Egger intercept test showed no pleiotropy (pintercept > 0.05) for these three obesity-related traits. In the leave-one-out test, we found that the MR estimates were stable when dropping a single SNP out, one by one in BMI and WC , p = 9.30 × 10–1] ,  × 10–1] .In this study, we performed MR analyses to test if human genetic evidence supported a causal effect of increased obesity-related factors  on the risk of IPF. Overall, the increased BMI, other than WC or WHR, showed causal effects on the risk of IPF. Our findings were further supported by replicated analysis and multivariable MR. Combined with a series of sensitivity analyses, the association between BMI and IPF risk can therefore be considered most reliable.2) with IPF who received a bilateral lung transplant may be at a higher risk of a 90-day mortality compared with patients of normal weight  and Asia and South America  .To the best of our knowledge, this study is the first in estimating obesity on IPF risk using MR methods. We applied a series of sensitivity analyses, as well as multivariable MR to minimize the influence of potential confounders and horizontal pleiotropy. There are also three major limitations in our study: we could not make a gender-stratification MR analysis for IPF because the author did not provide gender-specific data ; only EuIn summary, our MR results provided evidence that genetically determined BMI increment demonstrates a higher risk on IPF, expanding our knowledge of additionally novel etiology for IPF. Further studies are required to determine the reliability of BMI as a predictor of IPF risk, to evaluate the mediating mechanisms for potential intervention targets.


The custom functionalization of a graphene surface allows accessto engineered nanomaterials with improved colloidal stability andtailored specific properties, which are available to be employed ina wide range of applications ranging from materials to life science.The high surface area and their intrinsic physical and biologicalproperties make reduced graphene oxide and graphene oxide unique materialsfor the custom functionalization with bioactive molecules by exploitingdifferent surface chemistries. In this work, preparation  of reduced graphene oxide and graphene oxide derivatives functionalizedwith the well-known antibacterial agent salicylic acid is reported.The salicylic acid functionalities offered a stable colloidal dispersionand, in addition, homogeneous absorption on a sample of textile manufacture, as shown by a Raman spectroscopy study, thusproviding nanoengineered materials with significant antibacterialactivity toward different strains of microorganisms. Surprisingly,graphene surface functionalization also ensured resistance to detergentwashing treatments as verified on a model system using the quartzcrystal microbalance technique. Therefore, our findings paved theway for the development of antibacterial additives for cotton fabricsin the absence of metal components, thus limiting undesirable sideeffects. Antimicrobial and high-performance nanoengineered materials havebecome highly sought after. In this field, researchers mainly dealwith the identification of efficient methodologies for the customfunctionalization of material surfaces to provide enhanced colloidalstability and peculiar biological properties, with the developmentof reliable, eco-friendly, and scalable processes.1 and reduced graphene oxide (r-GO)2 showedsignificant antimicrobial properties toward a broad range of pathogens.4 These materials have also found application to produce antimicrobialcomposites,8 sometimes combined with well-known metal-based antimicrobial additivessuch as silver nanoparticles (AgNPs).11 However, for the latter,it has been demonstrated that the extensive use of AgNPs induces bacterialresistance limiting their scope as general antibacterial agents atlow concentrations, as they are used on fabrics.13In this framework, graphene oxide (GO)15 The wrapping of bacteria by graphene-based materials causes physicochemicalinteractions that give rise to perturbations of the bacterial membraneand deterioration of essential biomolecules .16 Similarly, these interactions isolate bacteriafrom the nutritive environment14 and inducereactive oxygen species (ROS)-dependent and ROS-independent oxidativestress.14 However, one main limitationin some applications of graphene-based materials as antimicrobialadditives is their tendency to form aggregates. Agglomeration weakenstheir dispersibility and adsorption ability, hence reducing graphene–bacteriaphysicochemical interactions.17 In this regard, a closerelationship exists between the graphene surface and antimicrobialefficacy; hence, graphene surface chemical functionalization has beenproposed as a valuable approach to prevent particle agglomerationand thus increasing antimicrobial activity.18 In particular, basal plane destruction and the covalent modulationof the graphene surface with oxygen-containing groups impact the antimicrobialactivity either by improving adsorption interactions  or enhancing ROS production.19 However, thescale-up of the functionalization process is the main drawback inhandling these materials and it limits industrial-driven applicationswhere either significant batches are required or the use of organicsolvents is avoided. Indeed, side aggregation phenomena of grapheneand reduced graphene flakes occur when concentrations higher than0.16 mg/mL in water and 60 mg/mL in N-methylpyrrolidone(for graphene) are reached.21On the other hand, despite the basis of antimicrobial mechanismsof graphene-based materials still being controversial, some scenarioshave been proposed leading to the identification of key insights helpfulfor the rational design of efficient graphene-based antimicrobialadditives and pointing out some of the issues that need to be addressed.1) and reduced graphene oxide functionalized with 4-aminosalicyclic acid(ASA) 3  3 1. ASA-3 4 and GO-SA 5  mimicking the cotton fabric in quartz crystal microbalance (QCM)experiments to assess the effect related to detergent treatments and(b) stabilizing the dispersion of the graphene materials and enhancingtheir interactions with the cotton fabric.In particular, we prepared the functionalized graphene-based materialsr-GO-SA  GO-SA 5 1, by expIt is worth noting that a quartz crystal microbalance (QCM-D) onthe CNC model system showed that the functionalization with the salicylicacid moiety significantly improved the physical interactions betweengraphene derivatives and cotton fibers even after surfactant solutiontreatments.Graphene oxide was purchased from NANESAas a spray dry powder GoNan  or as 0.4% w/w dispersion in water. Nanocrystalline cellulose(CNC) was purchased from Celluforce. All of the other reagents, forwhich synthesis is not described, were commercially available andhad been used without any further purification.Transmission electron microscopy(TEM): the materials were dispersed in Milli-Q water at 0.2 mg/mLconcentration and a volume of 5 μL was used for the analysis.UV–vis: the analysis was carried out on water dispersion at0.05 mg/mL unless otherwise indicated.1 (spray dry powder), 2.0 g of sodium deoxycholate , and 500 mL of Milli-Q water (concentration:1.0 mg/mL of 1). The resulting mixture was dispersedusing an ultrasonic bath . Then, sodium carbonate (Na2CO3) was added until a pH of 8, followed by 1.28mL of hydrazine solution (30% in water). The mixture was stirred at80 °C for 12 h. During the time of the reaction, the color ofthe dispersion turned from brownish to black. The resulting dispersionwas used for the preparation of r-GO-SA 4 (see below).An aliquot (10 mL) of the dispersion was dialyzed vs water and thenlyophilized to afford a black powder, which was used for the characterization.UV–vis: λmax = 271 nm . Fourier transforminfrared spectroscopy (FT-IR): 3610, 1695, 1513, 1185, and 1079 cm–1 . Elementalanalysis: C 45.19%, H 0.77%, N 0.83%. TEM images are provided .A 1L round bottomed flask was loaded with 500 mg of 1 (0.25 mg/mL) or substituting spray dry powder 1 witha cheaper (GO) water dispersion (0.4% w/w)) without appreciable differences.The same synthesis was performedwithout the use of SDC  and4-aminosalicylic acid  were dissolvedin 12.3 mL of a solution of NaOH (0.25% wt in water). The mixturewas added dropwise to an ice-cooled HCl solution  under vigorous stirring. The resulting solution was addeddropwise to a cooled dispersion of 2-SDC at pH 6 under vigorous stirring. The final dispersion was sonicatedfor 1 h (59 Hz) and then stirred at 80 °C for 12 h. The reactionmixture was filtered (0.4 μm polycarbonate membrane) and thefiltrate was dispersed in water (1.0 mg/mL) and dialyzed (cellulosemembrane cutoff = 14.000) vs water for 4 days to give 30 mg of purer-GO-SA 4. UV–vis: λmax = 210nm . TEM images are provided. Loading of salicylic acidbased on thermogravimetric analysis (TGA) under a nitrogen atmosphere4.70% w/w .Sodium nitrite (NaNO2 without the addition of SDC and r-GO 2 obtained fromthe reduction of a 0.4% w/w water dispersion of 1 (GO)without appreciable differences .The same procedure was also performed using r-GO 1 (spray dry powder) and 3 in a 1:1 weight ratio. A steelball of 1 cm diameter (26 g) was added and the jar was shaken for40 min at 25 Hz. The jar was washed with Milli-Q water to recoverthe material and to obtain a 1.0 mg/mL dispersion of GO-SA 5. The dispersion was dialyzed (14 kDa cutoff) until the UV–vissignal of 3 disappeared from the dialysis solution. Theprocess was repeated four times to obtain four samples for which theelemental analysis is reported in Table S1. UV–vis: λmax = 211, 261, and 299 nm . FT-IR: 3222, 1735, 1616, 1232,1074, and 781 cm–1 , TGA .A10 mL stainless-steel jar was filled with 200 mg of GO Procedure: Samples of woven fabrics(7 × 3 cm) were immersed in the dispersion of selected nanomaterialsand kept under stirring for 40 min. After that they were dried atroom temperature for 30 min and washed with ethanol.The followingdispersions of selected nanomaterials were used:2 and CNC: 2 0.3 mg/mL, CNC 0.5% w/w.Water dispersion of r-GO 4 (with and without SDC)and CNC: r-GO-SA 4 0.25 mg/mL, CNC 0.5% w/w.Water dispersion of r-GO-SA 1 and CNC: 1 0.25mg/mL, CNC 0.5% w/w.Water dispersion of GO 5 and CNC: 5 0.25 mg/mL, CNC 0.5% w/w. On a visual analysis, due to the simpledyeing technique, none of the sample is completely homogeneous, althougha higher homogeneity for fabrics dyed with functionalized GO and r-GOis evident .Water dispersion of GO-SA 2O2/water solutionin a ratio of 1:1:5 for 5 min at 75 °C, washed with Milli-Q waterand dried with nitrogen flux. After that a plasma cleaner was usedfor 10 min to completely oxidize the surface. Cellulose nanofilmson a sensor were prepared, as described by Gunnars and co-authors.29 First of all, an anchoring polymer (chitosan)was used to attach the cellulose onto the sensor, by dipping the sensorinto a dilute solution of the polymer (0.01 g/L). After 15 min, thesensor was washed in deionized water at the same pH of the polymersolution and dried in an oven at 55 °C for 15 min. The cellulose’sfilm was produced using microcrystalline cellulose (CNC) dissolvedin 50% wt N-methylmorpholine-oxide (NMMO) at 115°C. Dimethyl sulfoxide (DMSO) was then used to decrease the viscosity.A thin layer of cellulose solution was spin coated at 2500 rpm witha hot solution of NMMO-CNC on a prechitosan-coated gold sensor. Afterextensively washing with water, the sensors were left to dry overnightin the fume hood.QCM-D experiments were performed on a Q-Sense E1 instrument  equipped with a 1 flow liquid cell , containing a gold-coated quartz sensor with 4.95 MHz fundamentalresonance frequency, mounted horizontally. Prior to use, the sensorswere cleaned with ammonia/Hf) and corresponding energydissipation factors (D) were measured for the oddovertones (1st–13th). A stable baseline for both f and D of the different harmonics was ensured beforeinjection of the sample.The experiments were performed at 25 °Cand the solvent exchange in the measurement chamber was achieved witha peristaltic pump. First, the sensor was placed in the chamber andwater was injected at a low flow rate (0.07 mL/min), and the fundamentalresonance frequencies . The 532 nm laser line was used, in combinationwith an 1800 L/mm grating. The selected objective was ×50L (Leica).Spectra were acquired in the frequency range 102–3203 cmKlebsiella pneumoniae (ATCC 700603), Staphylococcus aureus (ATTC 29213), and Candida albicans (ATTC 90028). All of the isolates were retrieved from frozen glycerolstocks, streaked on a fresh Mueller–Hinton (MH) agar plate,incubated at 37 °C for 18 h and subcultured to provide freshcolonies.The antimicrobialactivity of the functionalized fabric pieces was determined againstATTC strains of 1,r-GO 2, r-GO-SA 4, and GO-SA 5 were dispersed in ultrapure distilled water to a final concentrationof 256 μg/mL and sonicated in a water bath sonicator for 30min before use. Broth microdilution susceptibility tests accordingto the European Committee on Antimicrobial Susceptibility Testing(EUCAST) international guidelines were performed. Tests were carriedout on a 24-well plate by adding 200 μL of the microbial cellsuspension equal to 5 × 105 CFU/mL. Scalar dilutionsbetween 128 and 2 μL/mL of each compound were added, and plateswere incubated overnight at 37 °C. MCC values were determinedby plating 5 μL of the content of each well on Mueller–Hiltonagar plates that were incubated for 24 h at 37 °C. The MCC aredefined as the lowest concentration corresponding to the death of99.9% or more of the initial inoculum. Each test was performed intriplicate and both negative and positive controls were included.The different solid compounds, GO 5 colony-formingunits (CFU)/mL of K. pneumoniae, S. aureus, and C. albicans.Fabric pieces of1 × 1 cm were individually contaminated with 20 μL of thesingle microbe suspension containing 2.5 × 10The seeded gussets were incubated at 37 °C overnightand then transferred onto solid nutritive Mueller–Hinton (MH)agar by placing the contaminated side in contact with the solid nutrientmedium for about 10 min. Then, each fabric was transferred into 500μL of MH culture broth in a 24-multiwell plate and incubated,together with the MH Petri dishes, overnight at 37 °C. Each fabricpiece was tested for sterility before the microbial contamination.Experiments were repeated in duplicate on different days.All contaminatedfabric pieces were investigated by SEM, (“Supra 24”Zeiss). Fixed and dried textiles were mounted onto an aluminum stubusing double-sided carbon tape and coated with a gold/palladium film(80:20) using a high-resolution sputter coater (Agar Scientific B7234).4 and GO-SA 5 isdescribed. These materials are structurally different in terms ofthe graphene platform employed and, accordingly, the type of covalentbond (C–C bond for r-GO-SA 4 and C–N bondfor GO-SA 5) between the graphene platform and the antibacterialagent. The synthetic process has been optimized in terms of reactionconditions  and graphene concentrationin the reaction mixture, paving the way for the scale-up of the process.Then, a significant degree of functionalization of the graphene platformhas been reached by exploiting classical solution synthesis for r-GO 2 and a mechanochemical process for GO 1.In 4 started with the reductionof 1 and the subsequent functionalization of the graphenereduced platform with salicylic acid moieties using 4-aminosalicyclicacid 3 in a Tour reaction. Similar approaches, usingbenzoic acid instead of salicylic acid residues, have been reported28 and they proceeded using exfoliated graphiteflakes in N-methylpyrrolidone with p-amino benzoic acid.30 In these works, the highest concentrationof graphene  was achieved at 0.125 mg/mL, thus strongly limiting the scale-upof the process. Alternatively, the use of sodium dodecylbenzenesulfonate(SDBS) as the surfactant has been reported allowing us to reach higherconcentrations (up to 0.4 mg/mL) of graphene during the functionalizationstep.31 However, the benzene ring of SDBSis itself prone to radical addition by aryl radicals produced duringthe reaction.32The synthesis of 1 with hydrazinehas been accomplished modifying a reduction protocol reported in theliterature,35 using sodium deoxycholate (SDC) instead of SDBS asthe surfactant, to avoid radical side reactions. The reaction wasmonitored by UV–vis spectroscopy and, as expected, the UV maximumshifted from λmax = 230 nm 37 to a λmax = 271 nm, which is usually associatedwith reduced graphene materials . The selected surfactant stabilized the resulting r-GO 2 dispersion in water at a concentration up to 1.0 mg/mL. An analyticalbatch of 2 was purified by dialysis (10 kDa membranecutoff) to remove SDC, lyophilized and then fully characterized . A transmission electron microscopy(TEM) analysis showed that 2 consisted mainly of monolayerflakes . The role of SDC wasconfirmed by the lower r-GO 2 concentration in the reactionmedium (0.25 mg/mL) obtained in a separate experiment, which was performedwithout the use of SDC. In addition, the overlapping of the UV–visspectra of the two batches of r-GO 2  confirmed that SDC only affected ther-GO concentration in the reaction medium. The experiments were performedusing the dry powder of GO but no apparent variation was observedusing the cheaper and commercially available GO dispersion (0.4% w/win water) . TheIR spectra  showedthe same main signals in the zone >3000 cm–1 dueto the presence of hydroxyl groups left after the reduction and themain band near 1550 cm–1. r-GO 2 wasthe substrate for the Tour reaction that was performed to ensure asignificant batch of functionalized nanomaterials in a congruent reactionvolume. Specifically, the preparation of the diazonium salt was carriedout modifying the protocol reported by Wei et al.28 Due to the low stability of the dispersion of 2 in acidic medium, the diazonium salt of 3 (5-fold excessin weight compared to 2) was prepared in a separate flaskand slowly added using a cannula in a cooled aqueous dispersion of 2 and SDC (see the 2 (pH 6) allowed the production of upto 500 mg of functionalized r-GO-SA 4 , and transmissionelectron microscopy (TEM) analysis . The UV spectrum  showeda new band (211 nm) due to the salicylic acid moiety linked to thegraphenic platform while the IR spectrum  did not show relevant differences resected from the startingmaterial r-GO 2 . The thermogravimetricanalysis  confirmed a goodloading of salicylic acid . It should benoted that to ensure no residual SDC affected the thermogravimetricoutcome, a control experiment, without SDC, was performed, and TGAanalyses of the two adducts were compared .In this work, the reduction of GO  see the . The mat-GO-SA 4 1 by main-GO-SA 4 . Then, t4 (1.0 mg/mL in water) was assessed by closely monitoring the absorbanceand hydrodynamic size over time . Data obtained showed that the UV absorbance slowly decreasedover the first 10 days by reaching a 0.5 mg/mL concentration thatremained stable for over 2 months .Both short- and long-term colloidal stability of -SA 5 was obtainedstarting from GO powder and 4-aminosalicylic acid 3 bymeans of a green and solvent-free mechanochemical process, exploitingthe nucleophilic substitution of the amino group of 3 on the epoxide groups on the GO platform. Specifically, the synthesisof 5 was based on a dry ball milling reaction of 1 and 3 in a 1:1 weight ratio and grinding ina 10 mL stainless-steel mixing mill with a 1 cm Ø stainless-steelsphere for 40 min at 25 Hz. The black powder was easily recoveredfrom the jar, and it was dispersed in Milli-Q water (1.0 mg/mL) anddialyzed (14 kDa cutoff) until no UV–vis signal of 3 was recovered in the dialysis solution. Then, 5 wasfully characterized by means of UV–vis and FT-IR spectroscopy. Notably, both thermogravimetricanalysis  and elementary analysis(Table S1) confirmed a significant loadingof salicylic acid, which shifted from 0.34 mmol/g of the Tour processup to 1.47 mmol/g in the ball milling process (4.70 vs 22.6% in weight).The functionalized GO1 and r-GO 2 onto model textiles isstill missing. We used a quartz crystal microbalance (QCM) to explorethe adsorption of GO 1, r-GO 2, and theirderivatives 4 and 5 onto crystalline nanocellulose(CNC) fibers, used as a model of cotton fibers, deposited onto a gold-coatedQCM sensor.Despite the numerous studiesthat make use of graphene and graphene oxide adsorbed onto textiles,a quantitative mechanistic study addressing the adsorption processof GO N-methylmorpholine-oxide (NMMO) at 115 °C.After extensive water rinsing, the sensor was left to dry overnightin the fume hood. The sensor was then placed in the measurement chamberand water was added at a low flow rate (0.07 mL/min); the fundamentalresonance frequencies (f) and the corresponding energydissipation factors (D) were measured for the oddovertones (1st–13th). A stable baseline for both f and D of the different harmonics was ensured beforeinjection of the sample. Then, 500 μL of a water dispersionof different samples  at a concentration of 0.05 mg/mL wasinjected in the measuring chamber  and D (ΔD) ofthe different harmonics. To check the stability of adsorption, thesamples’ injection and equilibration were followed by: (i)a first rinse with Milli-Q water  a step 2, 2,common step 2, 2, which f is related tothe mass variation40 while ΔD can be used for aqualitative profiling of structural changes in the system, in termsof viscoelastic properties of the film. A decrease in Δf implies mass addition to the sensor, whereas an increasein ΔD indicates that the system has becomeless compact.40In a QCM-D experiment, Δ2 dispersion, an almost complete desorption takesplace after the first rinse with Milli-Q water  results in the formation of a homogeneous layer acrossthe support, which is conserved after injection of the detergent andrinsing with Milli-Q water . Forr step 2, 2b, with  step 3, 2 and sub step 3, 2, pointi step 3, 2a. Conve1 , while forGO 1 the higher harmonics are more sensible to the massloading confirming that there is a translayer binding. Due to thepoor adsorption of r-GO 2 on the surface, as mentionedabove, no conclusions can be gathered in terms of depth profiling.In all of the measurements, we notice that the presence of salicylicresidues restricts the interaction of graphene derivatives closerto the surface  was used, and the ability of functionalizedgraphene materials 4 and 5 to be adsorbedonto the fabrics and thus, to provide antibacterial activity was evaluated.These fabrics (216 g/m2) are characterized by a plain weave.Cotton is the most widelyused natural fiber to produce fabrics that possess comfort, breathability,and low price. However, cotton textiles can easily store humidityand become a growth medium for microorganisms. For these reasons,significant efforts have been devoted to developing cotton treatmentsto confer antimicrobial properties to the fibers.4 or 5 (0.25 mg/mL) onto the cotton fabrics.CNC was used to optimize the adsorption of the materials, 1,2, 4, and 5, onto the fabrics. Thus, a fabricsample (7 × 3 cm) was immersed in a graphene dispersion  and left under stirring for 40 min. Then, cotton fabricswere dried at room temperature for 30 min, copiously rinsed with absoluteethanol and dried again at room temperature . Then, fabrics embedded with the antimicrobial fillerswere characterized by means of confocal Raman microscopy. Raman spectrahave been acquired directly on the textile, before and after the treatmentwith GO 1, r-GO 2, r-GO-SA 4, and GO-SA 5, to verify the absorption of samples andexplore their distribution within the fabric.Based on the results obtained from the model systems of cottonfibers, we used a simple protocol based on physical adsorption ofa CNC dispersion (0.5% w/v in water) of the graphene material 47 The D band (located near 1350 cm–1) resultsfrom the presence of vacancies and defects in the material. The nextband, the G peak, is related to the in-plane vibration of sp2 hybridized carbon atoms and is located near 1580 cm–1. The last peak (2D) is related to the number of graphene layersand is located near 2700 cm–1.The typical Raman spectrum of graphene-based materials containsthree main diagnostic bands, marked as D, G, and 2D.4 , can be unambiguously assignedto the G band of graphene, proving the effective adsorption of thegraphene derivatives on the fabric. The D band of graphene, clearlydetectable in the spectra of pure GO and r-GO, cannot be easily identifiedin –1, characteristic of the fabric. The 1597 cm–1 Ramanband, characterized by high intensity and unambiguously ascribed tothe nanostructured material, can act as a selective probe for mappingthe distribution of the graphene derivatives with and without salicylicresidues on the fabric. To this end, confocal Raman microscopy wasused in the area scan mode, to locate graphene derivatives over thefabric surface, after treatment with 4 and 5. Raman mappings were performed over the fabric’s surface,using a λmax = 532 nm incident wavelength. In themapping, Raman spectra were sequentially acquired from an array ofsample points spanning a 15 μm × 15 μm area of thetextile’s surface, with 2.5 μm spacing between points(see the SI for further details). The collectedspectra were analyzed to generate two-dimensional Raman images, wherethe color intensity at each pixel details the integrated G-band intensityin the range 1453–1669 cm–1.4 4a and GO4  reports a very similarbehavior, with r-GO-SA extensively covering the textile surface. Colormaps of fabrics dyed with 1 and 2 were alsoacquired as control samples , showing a similar distribution of samples over the fabric surface.ensities 5b and blensities 5c. The m4 and 5) and unfunctionalized graphene materials (1 and 2) were evaluated against strains of K. pneumoniae andS. aureus as representatives of, respectively, Gram negative (Gram (−))and Gram positive (Gram (+)) bacteria strains and C.albicans as representative of a fungus. Data obtained(Table S2) confirmed that all of the materialswere active at a 128–2.0 μg/mL concentration range. Particularly,r-GO-SA 4 showed the highest antimicrobial activity whencompared with other materials (Table S2). On the basis of these data, graphene-embedded fabrics (SI for details) for their antibacterialactivity against the same strains.Minimal Cytocidal Concentration of functionalized (obtainedS2 confir2O) dispersion had no inhibitory effect on microbial growth  , showedno activity (entry 4) toward either Gram (+) or Gram (−) bacteria.Conversely, the presence of SDC in the dispersion of r-GO-SA 4 (entry 5) ensured a protective effect against K. pneumoniae and only a few colonies of S. aureus have been observed as the readout of asignificant antimicrobial activity.Data showed that the fabric treated with a CNC  scanning electron microscopy (SEM) imagesof the graphene-embedded fabrics were recorded (To evaluate the alteration of the cell morphology of the threemicroorganisms (i.e., recorded 6.C albicans, S. aureus, and K.pneumoniae on fabrics. As expected, no evident alterationswere observed for all of the three microorganisms studied, as proofof the absence of cellular suffering. Conversely and according toresults obtained by in vitro antimicrobial analyses, all microorganismsshowed an altered morphology when grown on fabrics treated with eitherr-GO-SA 4 or GO-SA 5 (C. albicans cells (4 and GO-SA 5 also interfere with the growth of S. aureus on fabrics (K. pneumoniae appears clearly altered on fabrics treated with r-GO-SA 4 and GO-SA 5 ( GO-SA 5 6D–I. Spens cells 6F,I is s fabrics 6E,H and, GO-SA 5 6D,G withThe functionalization of reduced graphene oxide and graphene oxideis a useful approach for the preparation of metal-free antibacterialadditives that can be used directly on cotton fabrics. Reliability,eco-compatibility, and scaling-up of the process are key points whichonce addressed can pave the way for the translation of the use offunctionalized graphene materials from the bench to industrial-drivenapplication. In this context, we demonstrated that the functionalizationof structurally different graphene materials can be achieved by exploitinga gram-scale synthetic process which uses, accordingly with the protocolemployed, or water as the solvent in a reduced and optimized volumescale, or mechanochemical forces in an eco-friendly process. Despitethe presence of salicylic acid moieties on the graphene platform slightlyimproving the antibacterial activity, it surprisingly increased theinteractions of the graphene materials with the cotton fabric as demonstratedby a quartz microbalance study. Graphene versatility can be exploitedfor the development of new nanoengineered antibacterial cotton materialsfor a wide range of applications, including antimicrobial gowns inhealthcare settings.