Short-term exposure to ozone and nitrogen dioxide is a risk factor for acute exacerbation (AE) of idiopathic pulmonary fibrosis (AE-IPF). The comprehensive roles of exposure to fine particulate matter in AE-IPF remain unclear. We aim to investigate the association of short-term exposure to fine particulate matter with the incidence of AE-IPF and to determine the exposure-risk time window during 3 months before the diagnosis of AE-IPF.2.5). In the time-series data, we compared monthly mean exposure concentrations between months with AE (case months) and those without AE (control months). We used multilevel mixed-effects logistic regression models to consider individual and institutional-level variables, and also adjusted these models for several covariates, including temperature and humidity. An additional analysis with different monthly lag periods was conducted to determine the risk-exposure time window for 3 months before the diagnosis of AE-IPF.IPF patients were retrospectively identified from the nationwide registry in Japan. We conducted a case–control study to assess the correlation between AE-IPF incidence and short-term exposure to eight air pollutants, including particulate matter < 2.5 µm (PM2.5 30 days prior to AE diagnosis. Adjusted odds ratio (OR) with a 10 unit increase in NO was 1.46 [95% confidence interval (CI) 1.11–1.93], and PM2.5 was 2.56 (95% CI 1.27–5.15). Additional analysis revealed that AE-IPF was associated with exposure to NO during the lag periods lag 1, lag 2, lag 1–2, and lag 1–3, and PM2.5 during the lag periods lag 1 and lag 1–2.Overall, 152 patients with surgically diagnosed IPF were analyzed. AE-IPF was significantly associated with an increased mean exposure level of nitric oxide (NO) and PM2.5 is a risk factor for AE-IPF, and the risk-exposure time window related to AE-IPF may lie within 1–2 months before the AE diagnosis. Further investigation is needed on the novel findings regarding the exposure to NO and AE-IPF.Our results show that PMThe online version contains supplementary material available at 10.1186/s12931-021-01671-6. The nat(AE-IPF) . The two(AE-IPF) . Several(AE-IPF) , 8; howe3) and nitrogen dioxide (NO2) during 0–42 days prior to AE was shown in South Korea [3 was positively related to AE-IPF [Some patients with IPF experience severe deteriorations that are associated with rapid disease progression and high mortality, which is termed AE-IPF , 10. AE-th Korea . SimilarAmong ambient air pollutants, fine particulate matter is considered as particularly dangerous. Indeed, an association has been shown between the elevated levels of airborne fine particulate matter and the risk of hospital admissions for patients with asthma, chronic obstructive pulmonary disease (COPD) and cardiovascular disease –18. Dale2), nitric oxide (NO), NO2, nitrogen oxides (NOX), carbon monoxide (CO), O3, PM2.5, and PM10, were evaluated using the nationwide surgically diagnosed IPF and IIPs registry in Japan [We hypothesized that the short-term exposure to fine particulate matter could increase the incidence of AE-IPF. Therefore, we investigated whether the incidence of AE-IPF associated with increased mean exposure level of fine particulate matter in the month with AE. Furthermore, we performed an additional analysis with different monthly lag periods to determine the exposure-risk time window during 3 months before the diagnosis of AE. We also investigated the correlations between the AE of idiopathic interstitial pneumonias (AE-IIPs) and the air pollutant exposure levels. The exposure levels of eight air pollutants, namely sulfur dioxide (SOin Japan .We performed a case–control study to investigate the correlation between short-term exposure to air pollutants and the incidence of AE. A time-series data was used to compare monthly mean exposure concentrations between 41 months with AE (case months) and 5742 months without AE (control months)  witCO), HRCT pattern [We extracted the following information at the time of SLB from the database as baseline characteristics: age, sex, smoking status (pack-years), percentage of predicted value of forced vital capacity (FVC), percentage of predicted value of diffusing capacity of the lung for carbon monoxide . O3 measurements were obtained during the 15-h period of daylight (5:00–20:00). For the PM10 measurements, the measurements obtained as suspended particulate matter (SPM) from the website were used because SPM is defined as airborne particulate matter with a diameter smaller than or equal to 10 in Japan. Daily and monthly mean temperature and humidity values were obtained from the database of the Japan Meteorological Agency (https://www.data.jma.go.jp/gmd/risk/obsdl/index.php). We selected the air monitoring stations located nearest to the registered hospitals from 1907 air monitoring stations in Japan and obtained the levels of air pollutants, temperature, and humidity. The patients who developed AE were admitted to these registered hospitals. Demographic data for each prefecture and neighborhood-level factors in Japan were obtained from the 2015 national census (https://www.e-stat.go.jp/).Daily and monthly mean concentrations of SOMultilevel mixed-effects logistic regression models were used to evaluate the association between the incidence of AE-IPF and monthly mean exposure for each air pollutant by matching data on the case month with that on the control month. For each AE diagnosis, the case month was defined as 30 days before AE diagnosis  and cumulative exposure (lag 1–2 to lag 1–3). For example, lag 1 exposure refers to the exposure during 30 days prior to AE diagnosis, while lag 1–2 exposure refers to the exposure during 60 days before AE diagnosis. The definition of control and case periods in the additional analysis is shown in Additional file  to lag 3CO at the time of SLB (risk factors identified in previous studies [2 (ppb), NO (ppb), NO2 (ppb), NOX (ppb), CO (ppb), O3 (ppb), PM2.5 (µg/m3) and PM10 (µg/m3). We also assessed the association between the incidence of AE-IIPs and monthly mean exposure for each air pollutant using the same methodology used for AE-IPF. All analyses were conducted at a significant α level of 0.05. All statistical analyses were performed using the STATA 16.1 software . Complete details of the methods are available in the Additional file These models were adjusted for temperature, humidity, age, sex, smoking status (pack-years), the percentage of the predicted value of FVC and DL studies ), and ne studies . ResultsAmong the 465 patients included in the previous study , 113 wer2.5 was 2.56  (Table 2 (lag 2 and lag 1–2), PM2.5 (lag 1 and lag 1–2), and AE-IPF , NOX was 1.29 , and PM2.5 was 2.88  , NOX , PM2.5 , and AE-IIPs.  and PM2.5 and the incidence of AE-IPF. An increase in 10 µg/m3 of PM2.5 amplified the risk of AE-IPF by approximately 2.5-fold.Based on the nationwide surgically diagnosed IPF registry, the present study demonstrated that there was a positive relationship between short-term monthly exposure to PM3 and NO2 during 0–42 days prior to the diagnosis of AE-IPF [2.5 (France) or PM10 (South Korea) for AE-IPF [2.5 30 days before AE diagnosis has significant positive association with the increased risk of AE-IPF after adjusting for temperature, humidity, age, sex, smoking status, and percentage of the predicted value of FVC, DLCO, and neighborhood-level factors. On the other hand, no significant relationship was observed between AE-IPF incidence and exposure to NO2 and O3. The reason of the difference between our study and the previous studies on NO2 and O3 is unclear; however, the difference in the definition of IPF diagnosis, ethnicity, environment of studied areas and statistical methodology might explain that there was no relationship between these irritant pollutants and the incidence of AE-IPF in our study. In this study, the proportion of UIP pattern in HRCT was 15% of the IPF patients, which was less than that of the previous study [In the previous two studies (South Korea and France), a significant positive association was observed between the increased levels of Of AE-IPF . Moreover AE-IPF . Our stuus study . Future 2.5 level and the incidence of AE-IPF [2.5 tends to be deposited in the lower airways [2.5 triggers an inflammatory reaction and induces tissue damage in the lungs; thus, contributing significantly to AE-IPF.Several in-vivo and in-vitro findings support the biological plausibility of the correlation between the elevated PMf AE-IPF . PM2.5 t airways  inducing airways . Further airways –32. Ther2.5 levels and the AE-IPF diagnosis were observed during lag 1 and lag 1–2 months in our additional analysis. An increased mean level of PM2.5 during 15–21 days  showed the strongest impact on AE-IPF  [2.5 level in patients with IPF was 15.4  [2.5 level remains a possible risk factor of AE-IPF in Japan.The health benefits of pollution reduction strategies have been documented in the Asia–Pacific Region, including Japan . The Jap3 (2009) . In our r PM2.5) . Accordi2 during lag 3 was observed. The negative correlation between exposure to SO2 and AE-IPF has been unclear. Further research is needed to determine the clinical significance of these results.We demonstrated a novel correlation between short-term monthly exposure to NO and AE-IPF. There was no report that exposure to ambient NO is a risk factor for respiratory diseases, including AE-IPF, presumably because few countries measure ambient NO in the atmosphere. In addition, significant negative association between AE-IPF and exposure to SO2, NOX, and PM2.5. This is the first study to show the correlation between AE-IIPs and short-term exposure to ambient air pollutants. Further research is necessary to clarify the relationship between exposure to air pollutants, including fine particulate matter and AE-IIPs.This study establishes the significant positive correlations between the AE-IIPs and short-term monthly exposure levels of NO, NOThe strengths of the present study are as follows. First, accurate IPF diagnoses were obtained in our study. All patients with IIPs participated in this study were diagnosed using SLB samples and MDD using a new video-conferencing system were performed . AlthougSeveral limitations of this study should be acknowledged. First, this study was a retrospective study. Second, the sample size was relatively small and consisted only of Japanese patients; however, it comprised only patients with surgically diagnosed IPF, the most robust method for IPF diagnosis. Third, socioeconomic information that should be used for adjustment were not available in the database. Fourth, the addresses of patients were not available in the database; however, the hospital addresses were used as an alternative, because the addresses of patients are approximately 10 min away by ambulance from the nearby hospitals in Japan (Additional file 2.5 may be a contributing risk factor to AE-IPF. We identified that the exposure periods of ambient PM2.5 recorded between 1 to 2-month prior to AE diagnosis were positively associated with AE-IPF in the additional analysis. Consistent with previous reports, we confirmed that “the 0–42 days” period preceding diagnosis may be useful in evaluating the relationship between the mean level of airborne PM2.5 and incidence of AE-IPF. Although the recent reduction in the levels of air pollutants has conferred health benefits, further efforts are required to decrease exposure to PM2.5 and reduce the risk of AE-IPF in Japan. We also identified the positive association between short-term exposure to NO and AE-IPF. Prospective cohort studies are expected to validate the relationship between exposure to ambient PM2.5, NO, and AE-IPF.The results of the case–control study suggest that short-term monthly exposure to PMAdditional file 1. Supplementary Material.


Stenotrophomonas maltophilia K279a. We determined several crystal structures of L1 complexes with three different classes of β‐lactam antibiotics , with the inhibitor captopril and different metal ions . All hydrolyzed antibiotics and the inhibitor were found binding to two Zn+2 ions mainly through the opened lactam ring and some hydrophobic interactions with the binding pocket atoms. Without a metal ion, the active site is very similarly maintained as that of the native form with two Zn+2 ions, however, the protein does not bind the substrate moxalactam. When two Zn+2 ions were replaced with other metal ions, the same di‐metal scaffold was maintained and the added moxalactam was found hydrolyzed in the active site. Differential scanning fluorimetry and isothermal titration calorimetry were used to study thermodynamic properties of L1 MBL compared with New Deli Metallo‐β‐lactamase‐1 (NDM‐1). Both enzymes are significantly stabilized by Zn+2 and other divalent metals but showed different dependency. These studies also suggest that moxalactam and its hydrolyzed form may bind and dissociate with different kinetic modes with or without Zn+2 for each of L1 and NDM‐1. Our analysis implicates metal ions, in forming a distinct di‐metal scaffold, which is central to the enzyme stability, promiscuous substrate binding and versatile catalytic activity.Emergence of Enterobacteriaceae harboring metallo‐β‐lactamases (MBL) has raised global threats due to their broad antibiotic resistance profiles and the lack of effective inhibitors against them. We have been studied one of the emerging environmental MBL, the L1 from Stenotrophomonas maltophilia K279a has been studied by determining 3D structures of L1 enzyme in the complexes with several β‐lactam antibiotics and different divalent metals and characterizing its biochemical and ligand binding properties. We found that the two‐metal center in the active site is critical in the enzymatic process including antibiotics recognition and binding, which explains the enzyme's activity toward diverse antibiotic substrates. This study provides the critical information for understanding the ligand recognition and for advanced drug development.The L1 metallo‐β‐lactamase from an environmental multidrug‐resistant opportunistic pathogen  APSadvanced photon sourceCSGIDCenter for Structural Genomics of Infectious DiseasesDSFdifferential scanning fluorimetryEDTAethylenediaminetetraacetic acidIPTGd‐thiogalactosideisopropyl‐β‐ITCisothermal titration calorimetryKmMichaelis–Menten constantMBLmetallo‐β‐lactamaseMRmolecular replacementNDM‐1New Deli metallo‐β‐lactamase −1PCRpolymerase chain reactionRMSDroot mean square deviationSBCStructural Biology CenterTEVtobacco etching virus1Since penicillin was first used against bacterial infections, β‐lactam antibiotics have been used to treat a wide range of diseases and further advanced to several classes of drugs. These include compounds with a broad‐spectrum substrate profile such as penicillins, cephalosporins, monobactams, and carbapenems. The β‐lactam containing antibiotics are substrate analogs of bacterial transpeptidases involved in cell‐wall biosynthesis.Currently, β‐lactam‐based antibiotics cover more than 65% of all clinically applied antibiotics.+2, playing a key role in their catalytic activities, as such they are called metallo‐β‐lactamases (MBL). There are three subclasses for MBL proteins, B1, B2, and B3, on the basis of their protein sequence, substrate profiles and especially subtle but significant variations in their active site configurations for two Zn+2 ions.β‐lactam antibiotics are produced naturally by many bacteria and are used in interspecies competition against a broad spectrum of Gram‐negative and Gram‐positive bacteria. In response, a number of different β‐lactamase classes  and subclasses evolved and have been deployed by bacteria. Many of these systems have been identified and studied over the years. These functional studies are supported by numerous crystal structures, some determined at atomic resolution. β‐lactamases hydrolyze a four‐atom β‐lactam ring, abolishing its antibacterial properties. Enzymes belonging to classes A, C, and D β‐lactamases are serine‐based hydrolases.+2 scaffold, which anchors β‐lactam moiety of a broad range of substrates with different substituents.Substrate promiscuity of MBL stems from the MBL fold having a large, open binding pocket consisting of multiple flexible loops and a di‐nuclear Zn+2 ions to stabilize atoms in the β‐lactam ring. It is accepted that the Zn+2 ions are acting as a Lewis acid by interacting with the β‐lactam carbonyl oxygen and facilitating nucleophilic attack. The positive charge of metal ions offsets the negative charge developed on the oxygen atom in the tetrahedral intermediate anion. The zinc ion also lowers the pKa of the directly coordinated water molecule.The catalytic mechanism for MBLs has been studied greatly including L1, CcrA, BcII, and NDM‐1.Apparently, two zinc sites are not same: the first site Zn1, or M1 as a general metal site 1, is a tetradentate (three His residues and a water molecule for both B1 and B3 subclasses) in a tetrahedral configuration and the second site Zn2, or M2, is more variable penta‐ or hexa‐dentate  in an approximately trigonal bipyramid configuration. This subtle geometrical and chemical difference in MBL enzymesStenotrophomonas (Xanthomonas) maltophilia is an environmental multidrug‐resistant Gram‐negative bacillus found in aqueous habitat that is an emerging opportunistic global pathogen, particularly among hospitalized patients. S. maltophilia infections have been associated with high morbidity and mortality in severely immunocompromised and debilitated individuals such as cancer, cystic fibrosis, and transplant patients.S. maltophilia L1 is a subclass B3 MBL, one of two chromosome‐encoded β‐lactamases (L2 belongs to a class A).+2, Cd+2, and Cu+2). We focus here on the structural basis of how the L1 enzyme hydrolyzes different groups of lactam antibiotics and how the metal ions contribute to enzyme's activity. The roles of metals to the enzyme's biochemical function is analyzed and compared with more extensively characterized B1 class enzyme NDM‐1.22.11 space group. Each protein subunit makes extensive contacts with all three other subunits of the tetramer. Interestingly, all other L1 structures described here have only one protein chain in the asymmetric unit in the P6422 space group. The same tetramer is maintained with subunits related by the crystallographic two 2‐fold symmetries that are 120° apart. L1 is the only B class β‐lactamase (BBL) that forms a tetramer and the formation of a tetramer is critical for the catalytic activity and/or substrate profile of L1 as shown by mutational study.S. maltophilia K279a) used in this study.The native form structure (L1‐Native) adopts a tetramer of the well‐conserved αβ/βα MBL fold: as previously reported,2.2+2 (or Cd+2 or Cu+2) ions and the protein side chains coordinating metal ions at the bottom of the binding pocket. All the following atomic distances are observed in L1‐Native. His116 [standard BBL numberinghttp://firstglance.jmol.org/fg.htm?mol=5DPX, http://firstglance.jmol.org/fg.htm?mol=2FU8, http://firstglance.jmol.org/fg.htm?mol=5EVB, http://firstglance.jmol.org/fg.htm?mol=2FM6, http://firstglance.jmol.org/fg.htm?mol=2QDT, http://firstglance.jmol.org/fg.htm?mol=2AIO, and http://firstglance.jmol.org/fg.htm?mol=1SML) without ethylene glycol and with or without the other ligands , the Zn─X (N or O) distances are maintained normal from 2.0 to 2.2 Å. Therefore, it depends on the crystallization conditions and the presence of ligands, the location and the distance of the bridging water (or hydroxide) molecule, and the zinc configuration change. See Table As shown in the reported structures,Several studiesWhen the active site of L1 is compared to that of NDM‐1  were compared to L1  interactions are changed or replaced in the complexes. In all L1‐hydrolyzed antibiotic complex structures, the amine/imine nitrogen of the 5‐ or 6‐membered ring interacts with Zn2 (or M2) and both oxygen of the carboxyl group that attached to the same ring interact with Zn2 (or M2) and OG atoms of Ser221 and Ser223. The carbonyl moiety attached to the lactam ring becomes a carboxyl or carbonyl group dependent on the local condition. Except for the L1‐Mero complex, the bridging water (or hydroxide) molecules are maintained in the same positions. There are weak long‐range hydrophobic interactions between hydrolyzed antibiotics and protein residues in the pocket such as Trp39, Phe156, Ile162, and Pro226. Some of the extended parts of lactam antibiotics, away from the opened lactam ring, which contacts a metal directly, are disordered and not well‐defined   are better defined. In the NDM‐1 structure, Trp93 and Met63 provide hydrophobic interactions for the phenyl group of penicillins. However, the phenyl group of penicillin G in L1‐PenG, if it were to follow a similar orientation and location to that in the NDM‐1 complex, can be in position to clash with the side chain of Tyr32. Instead, it orients upward and gets exposed to solvent, which may lead to disordering. Although NDM‐1 has higher Km values (~200–300 μM) for penicillins (penicillin G and ampicillin) than L1 (~40–50 μM) (see Table ) Figure e: OveralL1‐Imip and L1‐Mero (Carbapenems)  of the hydrolyzed meropenem (hydrolyzed by L1 and NDM‐1) have similar conformations with the carboxyl oxygen replacing the bridging water molecules. One exception is in the VIM‐1‐hydrolyzed meropenem structure (PDB ID http://firstglance.jmol.org/fg.htm?mol=5N5I), where the carboxyl group rotates about 60° counter clockwise (CCW) along the C5–C6  has the same conformation. However, other hydrolyzed imipenem , structures with NDM‐1 adopted the conformation of the hydrolyzed meropenem. Both pyrrolines of hydrolyzed imipenem and meropenem seem to adopt a similar Δ1β tautomer) Figure d. All reL1‐Mox  ,) Figure f: The L12.6http://firstglance.jmol.org/fg.htm?mol=2FU8, http://firstglance.jmol.org/fg.htm?mol=5ZJ2, http://firstglance.jmol.org/fg.htm?mol=4EXS, and http://firstglance.jmol.org/fg.htm?mol=4C1C)D‐ or L‐captopril has been one of the most popular starting thiol‐based inhibitors for metalloenzymes as it can form a specific ternary complex enzyme‐metal‐inhibitor, which provides better selectivity toward drug targets than those of metal‐stripping type inhibitors Figure c. In the2.72). The active site conformation was quite similar to that of the structures with the metals and with bound ligands , isothermal titration calorimetry (ITC), and enzyme assays in comparison with NDM‐1.2.8.12+, Cd2+, Cu2+, Co2+, Ni2+ and Mn2+, as well as Mg2+, as a control  of Zn2+, NDM‐1 melts at 15°C higher indicating that the enzyme is much more thermally stable. The melting curve indicates a very small additional melting point at 46°C, which suggests that one zinc ion might leave at the temperature but the protein may still maintain its integrity. Other metals showed the different degrees of melting point shifts but all showed lower melting temperature (Tm) than zinc and each curve indicates only one melting point. It is shown larger shifts in the order of Zn (15°C) > Cd (10°C) > Co (7°C) > Ni (5°C)  required for MBLs for their substrate binding and optimum catalytic activity, the natural metal that appears most is zinc. However, several metals were reported to substitute zinc with variable catalytic activity. For example, cadmium ions have shown to have significantly reduced activity for NDM‐1.2.8.22+ and Mn2+, a similar progression is observed. It is known that some metal ions can replace zinc ions with a comparable catalytic efficiency. Perhaps, when zinc ions are depleted by EDTA, the protein may undergo some conformational changes, albeit subtle, and it may take some time to recover, presumably in a metal‐specific manner, even with an excess amount of metal ions. After EDTA‐treatment, both proteins may have the conformation in which the canonical metal zinc can bind without much change (conformation and energy). Thus, the recovery with zinc was fast but not with other metals.Our DSF analysis has shown that different metals  stabilize the enzymes differently. Enzymatic activities are observed for the same metals used for DSF analysis to learn how well these metals replace zinc ions. The metal‐dependent enzymatic activity of L1 and NDM‐1 is characterized by one of the carbapenems, meropenem, which has a characteristic absorbance near 300 nm Figure c. In the2.8.3The structural information from the EDTA‐treated crystals indicated that metal ions are required for specific ligand binding. To better understand the dynamic properties of ligand binding by L1 and NDM‐1 in comparison, the enzymes treated with EDTA to remove most of the zinc ions in the protein were titrated with moxalactam using ITC. NDM‐1 and L1 also show the distinct kinetic profiles in the presence and absence of zinc ions as revealed in the ITC analysis3S. maltophilia and compare it to properties of class B1 NDM‐1 MBL.In this study, we report structural, thermodynamic, and enzymatic analyses of the class B3 L1 MBL from +2 ions or the other metal ions that replace zinc. Only a few residues of the protein, Tyr32, Ser221, and Ser223, are involved in the direct interaction with ligands. The protein provides a large binding space to accommodate a broad range of β‐lactam antibiotics, which bind directly to the di‐metal scaffold formed at the bottom of the pocket in the enzyme. This di‐metal scaffold with Zn+2 ions  mainly carries out the catalysis and no protein atom other than side chains forming the di‐metal scaffold participates directly in the catalytic reaction.The structures of L1 in complexes with hydrolyzed antibiotics and the inhibitor captopril with zinc or other metals showed that there is no significant structural difference between the native form and the complexes with ligands and with different metals. Most of the direct contacts made by the ligands are to two Zn+2 ions was produced by treating crystals with EDTA. Analysis of the structures from the EDTA‐treated crystals, apo form, complexes with moxalactam, and different metals has shown that all structures maintained much of the same conformation as those of structures from untreated crystals. The L1 structures from EDTA‐treated crystals, soaked in moxalactam solution without any metal, did not contain a ligand, thus supporting the notion previously suggested by the biochemical study for B3 MBL GOB‐18 that the functional metal scaffold is required not only for catalysis but also for β‐lactam binding.The L1‐structure without ZnBiochemical analysis by DSF assay has shown that metal ions increase stability to both L1 and NDM‐1. However, NDM‐1 and L1 have different stability profiles with different metals. Zinc is most favorable for NDM‐1's stability and other metals provide varying degrees of stability. For L1, many metals including zinc provide similarly high degrees of stability. Kinetic analysis by ITCOur study suggests that the metal scaffold is central to MBL's functional activity, contributing to stability, substrate binding, and catalysis. L1 of subclass B3 MBL and the B1 MBL enzyme NDM‐1 have similar but distinct metal scaffolds, which contribute to differences in substrate binding and catalysis by the MBL.Our study implicates that MBLs of different subclasses have subtly different di‐metal scaffolds, which may confer diversity in substrate binding and catalysis.44.1S. maltophilia K279a was amplified from the genomic DNA with KOD Hot Start DNA polymerase. For the construct, 5′ TACTTCCAATCCAATGCCAGCGCCGCCGAGGCAC forward and 5′ TTATCCACTTCCAATGTTAGCGGGTCCCGGCCGTTT reverse primers were used. The PCR products were purified and cloned into the pMCSG53 according to the ligation‐independent procedureE. coli BL21(DE3)‐Gold strain (Stratagene). A single colony was picked, grown, and induced with isopropyl‐β‐d‐thiogalactoside (IPTG). The cell lysate was analyzed for the presence of the protein with the correct molecular weight. The solubility was analyzed via small‐scale Ni‐NTA affinity purification and overnight tobacco etching virus (TEV) protease cleavage followed by centrifugation at 3,200g of digested sample. For large‐scale purification, a fresh transformation was made into the E. coli BL21‐Gold (DE3) strain.The gene cloning was performed according to Kim et al.4.2600 ~1.0, the culture was cooled down to 4°C and 10 ml of 4 M K2HPO4 was added. Protein expression was induced overnight at 18°C with 0.5 mM IPTG. Bacterial cells were harvested by centrifugation at 7,000g  and cell pellets were resuspended in a 35 ml lysis buffer  with protease inhibitor cocktail  per liter culture and treated with lysozyme (1 mg/ml) and sonication. Debris was removed by centrifugation. L1 protein was purified via Ni affinity chromatography using the AKTAxpress system . The column was washed with 20 mM imidazole (lysis buffer) and eluted in the same buffer containing 250 mM imidazole. Immediately after purification, the His‐tag was cleaved at 4°C for 20 hr using a recombinant His‐tagged TEV protease, resulting in an untagged protein with an N‐terminal Ser‐Asn‐Ala peptide. An additional Ni affinity chromatography purification was performed to remove the protease, uncut protein, and affinity tag. The purified protein was then dialyzed against a 15 mM HEPES pH 7.0, 100 mM NaCl, and 2 mM TCEP buffer.For purification of the protein used for crystallization, a 1 L culture of enriched LB medium was grown at 37°C (190 rpm). At ODThe protein concentration was estimated by UV absorption spectroscopy (280 nm) using a NanoDrop 1000 spectrophotometer (Thermo Scientific). The purified L1 was concentrated using a centrifugal filter  to 71 mg/ml.4.31, which appeared within a week in condition #77 (G5) of the MSCG‐4 containing 0.1 M HEPES pH 7.5, 10% (w/v) PEG 4000. Since no ligands would co‐crystallize, all crystals of the complexes were prepared by soaking the native form crystals in the same crystallization solution with the ligands. For example, L1‐PenG crystals were prepared from the crystals grown in the condition Index A4  by being soaked in the same solution plus 5 mM penicillin G. Similarly, L1‐Capt crystals were prepared from the Peg‐Ion condition F4  with 25 mM captopril. L1‐Imip crystals were produced from the Peg‐Ion condition E8  with 5 mM imipenem, L1‐Mero crystals were prepared from the Index condition H2  with 5 mM meropenem, and L1‐Mox crystals were from soaking in Index H10  with 5 mM moxalactam. Prior to flash freezing in liquid nitrogen, crystals were cryo‐protected by briefly soaking in the crystallization solution containing up to 25% ethylene glycol.Initial crystallization screening was done with the sitting‐drop, vapor‐diffusion technique in 96‐well CrystalQuick plates (Greiner Bio‐one) using a Mosquito robot (TTP LabTech) and commercial crystallization kits including MCSG 1‐4 (Anatrace), Index (Hampton Research), and Peg‐ion (Hampton Research). The protein concentration used was 15 mg/ml. The L1 protein construct used for this study crystallizes readily in several different conditions in the presence of zinc ions. Among many diffraction quality crystals, the best crystals of the native form (L1‐Native) were monoclinic P22 followed by another soaking with 10 mM moxalactam (L1‐Cd‐Mox‐E), or (c) soaked in CuCl2 followed by another soaking with 20 mM moxalactam (L1‐Cu‐Mox‐E). All soakings were done for 10 min.Crystals of L1‐E, L1‐Cd‐Mox‐E, and L1‐Cu‐Mox‐E were prepared from the crystals produced in the condition PEG‐Ion E8. For L1‐E, the crystals were soaked in the same well solution additionally containing 5 mM EDTA for up to 48 hr. After given times, crystals were washed briefly (30–45 s) three times in the cryo‐solution, which contains an additional 25% ethylene glycol to remove most of the EDTA on the crystals, checked for diffraction, and used for data collection and structure determination. For L1‐Cd‐Mox‐E and L1‐Cu‐Mox‐E, the crystals were soaked for 48 hr with 5 mM EDTA and then washed with the same well solution (without EDTA) three times over 5 min, then (a) soaked in the solution containing 10 mM moxalactam and checked for diffraction or (b) soaked in 10 mM CdCl4.4ω for 90–305°. Data for the crystals of L1‐native (up to 1.70 Å resolution) and L1‐Capt (1.40 Å) were obtained at SBC‐19ID beamline on the dectris Pilatus3X 6M detector, and those of L1‐Mox (1.52 Å), L1‐PenG (1.65 Å), L1‐Imip (1.90 Å), L1‐Mero (1.98 Å) were collected at SBC‐19BM. They were recorded on the ADSC Q210r detector both with an X‐ray wavelength near 12.66 keV (0.97927 Å) for molecular replacement (MR) phasing . Similarly, the data for EDTA‐soaked crystals  and for the crystals of the EDTA‐soaked then treated with additional metal and antibiotic soaking  were collected. All diffraction images were processed using the HKL 3000 suiteThe crystal diffractions were measured at a temperature of 100 K using either a 0.5 s exposure per 0.5° rotation or a 1 s exposure per 1° rotation on 4.5http://firstglance.jmol.org/fg.htm?mol=5DPX) as the search model, and the rest of the structures were determined by using the L1‐Native structure as the search model. The initial models were manually adjusted using COOTThe structures were determined by MR using molrep implemented in the HKL3000 software package.Rwork and Rfree with regards to each resolution limit of all corresponding data. The stereochemistry of the structures was checked with PROCHECKRwork/Rfree of 17.6/19.8% includes four protein chains, each containing two zinc ions, and a total of nine ethylene glycol and 660 water molecules in the asymmetric unit. Similarly, L1‐Capt structure has one protein chain, two zinc ions, two captopril molecules, three formate molecules, one ethylene glycol and 291 water molecules; L1‐Mox has one protein chain, two zinc ions, a hydrolyzed moxalactam, three ethylene glycol molecules, and 290 water molecules; L1‐PenG has one protein chain, two zinc ions, a hydrolyzed penicillin G, a succinate, a bis‐tris, a sulfate, polyethylene glycol (PEG), one additional zinc ion and 226 water molecules; L1‐Imip has one protein chain, two zinc ions, a hydrolyzed imipenem, and 222 water molecules; L1‐Mero has one protein chain, two zinc ions, a hydrolyzed meropenem, a PEG, an ethylene glycol, and 149 water molecules. Ethylene glycol, PEG, sulfate, succinate, and bis‐tris molecules are originated from crystallization and/or cryo‐solution.The final structures converged to reasonable The structures of the metal‐free form and the complex with cadmium or copper ions were determined and refined by the same procedure. The final L1‐E structure (1.80 Å) includes the protein chain, one ethylene glycol molecule, and 107 water molecules. The L1‐Cd‐Mox‐E (1.60 Å) structure contains the protein chain, hydrolyzed moxalactam, two cadmium ions, and 273 water molecules; and L1‐Cu‐Mox‐E (2.38 Å) contains the protein chain, hydrolyzed moxalactam, two copper ions, one ethylene glycol molecule, and 52 water molecules. Coordinates of the structures have been deposited in the Protein Data Bank and the PDB IDs are shown in Table 4.62, CdCl2, MgCl2, NiCl2, CoCl2, CuCl2, and MnCl2) and SYPRO Orange dye (Invitrogen) were added in a buffer 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM TCEP. DSF of 50 μl samples on a 96‐well PCR plate (BioRad) was done on CFX Connect Real‐Time System (BioRad). Software parameters were lid temperature 105°C, sample equilibration 20°C for 30 min, and melting curve fluorescence signal acquisition from 25 to 90°C with ramp rate 1°C per 60 s. The experiment was done in triplicate and the resulting numbers were averaged.Samples of NDM‐1 and L1 were diluted to 250 μM in a buffer containing 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM TCEP. Zinc ions were removed from the protein samples by incubating with 5 mM EDTA for 30 min and then dialyzing three times over 16 hr at 4°C using the buffer solution of 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM TCEP. For the experiment, to 10 μM each of the metal‐depleted NDM‐1 or L1, 100 or 500 μM selected metals  with Origin7 and VPViewer 2000 software at 25°C with the reference power of 15 μcal/s. For the control, the buffer solution without antibiotics was used. The ITC experiment was carried out in single injection mode with injection parameters of volume 42.5 μl, duration 300 s, and spacing 1800 s. After injection, the molar ratio between protein and moxalactam was 1:2. The experiment was done in triplicate and the resulting numbers were averaged.For the ITC assay, 15 μM each of NDM‐1 and L1 in 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM TCEP buffer were prepared with 2 mM ZnCl4.82, CdCl2, MgCl2, NiCl2, CoCl2, CuCl2, and MnCl2). Each enzyme + metal sample was 10‐fold diluted into 0.3 mM meropenem  in the buffer containing 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM TCEP, and 10 μM EDTA. After 1 min of incubation, absorbance was measured using NanoDrop 1000 (Thermo Scientific) spectrophotometer. The experiment was done in triplicate and the resulting numbers were averaged.To study the activity of purified MBLs in the presence of different metals ions, 400 μM protein samples of NDM‐1 and L1 were treated with 5 mM EDTA for 2 hr. The EDTA‐treated protein samples were diluted to 2 μM in a buffer containing 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM TCEP, then incubated for 20 min with 250 μM of each selected metal (ZnCl


Direct interaction between nanocrystals and graphene results in quenching of up-conversion emission and shortening of luminescence decay times, due to the energy transfer to graphene. The amount of the energy absorbed by graphene can be enhanced by coupling Er3+/Yb3+ doped up-conversion nanocrystals with silver nanowires. Microscopy studies with high spatial resolution together with time-resolved analysis of nanocrystal luminescence show increase of the emission rates with fourfold enhancement of the intensity for nanocrystals placed in the vicinity of silver nanowires. This strong enhancement emerges despite simultaneous interaction with graphene. The hybrid nanostructure provides thus a way to combine optical activity of up-conversion nanocrystals and enhancement provided by metallic nanowires with excellent electrical and mechanical properties of graphene.Single nanocrystal spectroscopy is employed to demonstrate metal-enhanced optical response of Er From the point of view of optical activity, a single layer of graphene absorbs 2.3% of incident light, independently of the wavelength, in the spectral range from 400 to 750 nm2. Although the zero-energy gap of graphene inhibits in principle any photoemission associated with radiative recombination of charged carriers, under specific circumstances, visible emission upon excitation with ultrashort laser pulses has been demonstrated3. Recently, an important body of new effects and applications is focused on hybrid nanostructures, combining unique properties of graphene and various nanoemitters.Graphene, a monolayer of carbon atoms arranged into a two-dimensional hexagonal lattice, which exhibits high crystal quality, is well known for its remarkable thermal, mechanical, and electrical properties7. However, weak optical activity of graphene, including absorption and absence of band-to-band emission, makes it not very useful for strictly optical applications. To overcome this problem the concept of optical antennas (sensitizer) coupled with graphene has been postulated8. For instance, in the graphene-quantum dots coupled system, amount of energy absorbed by the antenna and efficiently transferred to graphene exceeds absorption possibilities of graphene itself9. It was shown that efficiency of the energy transfer depends also on the emission wavelength and on the number of graphene layers10. In these experiments graphene was recognized to be a perfect acceptor. For this reason, emission of the sensitizer within such a hybrid system is very strongly disrupted or can even be completely quenched11.Indeed, combination of functional molecules with excellent electrical properties of graphene opens possibility for new applications in sensorics and optoelectronics14. Due to unique optical properties they have found number of applications in lasers15, displays17, bio-medical contrasts/markers19 and sensors20. Recently, their application palette has been significantly extended due to incorporating interactions with graphene25. Herein, we use up-conversion NCs to sensitize sequential 2-photon absorption and introduce emissivity to the graphene-based hybrid system.In this paper, we construct an advanced nanostructured material, which combines physico-chemical functionalities with optical and plasmonic properties, and experimentally study its capability for optimized optical response. The main components for engineering the novel range of functionalities of luminescent up-conversion nanoparticles prepared by solution wet chemistry methods concern combination of energy transfer and plasmon enhancement. In particular, we present a new type of three-component hybrid nanostructure consisting of graphene layer combined with rare-earth co-doped nanocrystals (NCs) and silver nanowires (NWs). Rare-earth doped materials are known for their outstanding optical properties, utilizing Stokes and anti-Stokes (up-conversion) luminescence occurring in the visible and infrared spectral ranges28. Due to their elongated shapes and lengths in the range of tens of micrometers, they facilitate transport of the energy through surface plasmon polaritons (SPPs), which has been considered in the context of applications in sensorics and integrated optoelectronic circuits32. Herein, we use silver nanowires to enhance efficiency of the NCs absorption/emission, and to compensate luminescence quenching that is usually observed in the presence of graphene11. Consequently, the three-component hybrid nanostructure features bright luminescence despite the interaction with graphene. We show that under certain conditions it is possible to observe simultaneous interactions between all three ingredients  within the nanostructure. The optical properties of such a system, which bridges all characteristic features of its components, i.e. spectrally broad absorption, up-conversion luminescence, sensitivity to light polarization, can be accompanied by excellent electrical conductivity and mechanical strength of graphene. Appropriate tailoring of the nanohybrid structure not only minimizes any interference between functionalities, but can also either bring synergy between them or impart a new property to the advance nanomaterials. Due to this combination, and the additional functionalities resulting from interactions between its components, the described hybrid nanostructure may find applications in sensorics and optoelectronics devices.On the other hand, silver nanowires are known to enhance both Stokes and anti-Stokes luminescence of various emitters due to antenna and Purcell effects33. In this process, graphene is grown on a copper substrate and then it is covered by a polymer layer. In the next step, the metallic substrate is etched chemically, and graphene is transferred on a glass coverslip. In the final step, the stabilizing polymer is dissolved in a suitable solvent. High quality graphene substrates used in this work were purchased in ACS materials.The hybrid nanostructure studied in this work is assembled of three components: a layer of graphene, rare-earth doped nanocrystals, and silver nanowires. High quality monolayer graphene was prepared by chemical vapor deposition, as described elsewhere34. Their lengths vary from 10 to 15 µm, while their diameters are of the order of 100 nm. Extinction spectrum of Ag nanowires  and Yb3+ (20%) ions, synthesized as described previously36. Transmission electron microscopy images indicate that the NCs are of quasi-spherical shape, with typical diameters of about 30 nm  containing silver nanowires previously deposited on the graphene layer. Because graphene covers only part of the substrate, both isolated NCs, NWs, and coupled NCs-NW nanostructures deposited on graphene or glass can be accessible within one sample . For excitation we used continuous-wave/pulsed fiber coupled 10 mW single mode laser diode, operating at 980 nm. The laser beam is formed into a Gaussian beam, which allows to achieve excitation spot of about 450 nm in a diameter. Photoluminescence (PL) of NCs was detected using single photon counting module  coupled with multifunctional counter card . The images were acquired using band pass optical filters: 550/40 nm and 650/40 nm, dedicated for green and red emission of NaYF4:Er3+/Yb3+ nanocrystals, respectively. Time-resolved measurements (luminescence transients) were collected by a multiscaler card  with resolution about 1 µs, while the laser was triggered by a programmable functional generator (Keithley 3390). The emission spectra were recorded with Czerny-Turner monochromator , coupled with CCD camera , and operating with the spectral resolution better than 1 nm.The optical properties of the hybrid nanostructure were examined using confocal luminescence microscopy and spectroscopy. The experimental setup was based on a Ti-S microscope body (Nikon). The sample was mounted on a piezoelectric stage , allowing for raster-scanning along both 26.The functionality of the hybrid nanostructure composed of three components: up-converting nanocrystals, silver nanowires and graphene, is determined by interactions that occur between the components in close proximity. As graphene is an efficient acceptor of energy, it is expected that upon excitation of the up-converting nanocrystals, the energy will be dissipated as heat in the graphene layer via non-radiative energy transfer. The absence of any luminescence from graphene implies that the energy transfer can be quantified exclusively by measuring the emission intensities and lifetimes of the NCs emission. On the other hand, placing the NCs in the vicinity of silver nanowires, which exhibit strong plasmon resonance, leads to enhancement of up-converted emission. As shown previously, the enhancement measured in such a structure originates from the combination of increase of the absorption and excitation rates (the Purcell effect) associated with the shortening of luminescence lifetime38. Rough comparison of the images obtained for graphene and glass substrates indicates that the intensities measured for the up-converting NCs deposited on graphene are considerably lower, presumably due to the non-radiative energy transfer from NCs to graphene. In addition, we notice twice as large surface density of nanocrystals on graphene with respect to the glass substrate, which might be due to better surface adhesion of the colloid to the graphene.In Fig. 37. Indeed, assuming homogeneous distribution of dopant ions within NCs and spherical shape thereof, seven-fold increase of the emission intensity translates into almost two-fold increase of the diameter. This simple calculations are consistent with TEM data, where observed diameters of the NCs vary from about 20–40 nm  of about 40 kcps for both emission bands.Statistical distributions of the emission intensity obtained for single nanocrystals deposited on glass and graphene are presented in Fig.  nm Fig. b, c. The−4)40, we can expect that the presence of graphene substrate affects most of the ions even within the largest (thus brightest) NCs, modifying their emission intensity42. On the other hand, the nonradiative energy transfer from NCs to graphene improves the amount of energy absorbed by the graphene itself. In other words, NCs sensitize graphene for IR sequential absorption of two photons.In the case of NCs deposited on graphene, the intensities range from 5 to 25 kcps for both emission bands, and are roughly 2 times lower than for NCs on glass, as can be seen in Fig. 11, introducing plasmonically active metallic nanostructures may circumvent this effect, leading to a structure which remains optically active. The maps displayed in Fig. 27. Importantly, the positions of silver nanowires can be precisely determined using a backscattering intensity maps shown in Fig. While interactions between nanocrystals and graphene lead to significant reduction of the emission intensity, as reported also by others26. At the same time, the distribution of intensities is substantially broader compared to the reference. As metal enhanced luminescence is strongly dependent on the distance between an emitter and a metallic nanoparticle, variation of the distance directly translates into the variation of the emission intensity38.Interaction between up-converting nanocrystals and silver nanowires strongly influences the distribution of emission intensity of the former. The results of statistical analysis presented in Fig. Depositing both up-converting nanocrystals and silver nanowires on a graphene substrate turns on another interaction, namely, the energy transfer from the nanocrystals to graphene. It occurs for nanocrystals deposited and localized both in close to silver nanowires and in some distance from them. Exemplary luminescence intensity map is presented in Fig. Experiments carried out in the steady-state mode, while providing a way to compare the intensities and thus enabling estimation of the emission enhancement, fall short in regard to assess whether enhanced absorption or emission rates are responsible for this enhancement. It is also difficult to conclusively demonstrate and quantify interactions of NCs with graphene, as well as NCs and NWs with graphene in the three-component hybrid nanostructure. These questions can be answered by measuring luminescence decays of individual nanocrystals. Again, due to the absence of graphene photoluminescence, we focused solely on determining the optical response of the sensitizers .21. The origin of this behavior is related to spatial distribution of Er3+ ions within nanocrystals, resulting in variation of distances between particular ions and nanowire and/or graphene. In general, we observe monotonic shortening of emission lifetimes in the direction from NCs coupled with NWs (on glass), through those coupled with graphene, till the NCs coupled with NWs on graphene. As displayed in Fig. 3+ ions, localized closer and further from the surface of the nanocrystal. In order to simplify the analysis, we extracted decay parameters using the approach based on amplitude-weighted average decay time27. The results of this analysis are summarized in Fig. In Fig. 3+ ions) to graphene monolayer. Interestingly, very similar values of the decay times we extracted for NCs coupled with NWs on glass. The average lifetimes are reduced to 65 μs for green and 130 μs red emission  is about 100 μs for green, and 180 μs for red emission Fig. a,b. For ion Fig. c,d.According to acquired data we noticed inhomogeneous character of the samples. This is due to inevitable distribution of nanocrystals sizes, nanowires diameters/lengths, as well as random deposition of these nanostructures on the glass/graphene surface. Random distances between interacting objects strongly determine strength of the interactions, what can blur the image of physical processes taking place within considered system. For this reason, to draw more universal conclusions, we accomplished statistical analysis of estimated lifetimes and presented in histograms in Fig. We observed quantitatively new effects for three-component sample. Since it consists of NCs, NWs and graphene, one would expect much more complex interactions occurring between its ingredients than already discuss. As presented in Fig. In this work we comprehensively describe the optical properties of a three-component hybrid nanostructure consisting of graphene, up-conversion nanocrystals and silver nanowires. For nanocrystals on graphene, where the energy transfer is dominant, we observe decrease of the emission intensity accompanied by shortening of luminescence decay times. The results of single nanocrystal spectroscopy prove that introducing silver nanowires yields strong increase of quantum efficiency and thus the emission intensity, despite the quenching due to the energy transfer. Indeed, fourfold intensity enhancement for single nanocrystals coupled with silver nanowires can be achieved. Such a strong enhancement emerges despite simultaneous interaction with graphene. Importantly, the acquired luminescence decay profiles confirm that nanocrystals can simultaneously interact with both graphene and silver nanowires. The hybrid nanostructure described in this work provides a way to combine optical activity of up-conversion nanocrystals and enhancement provided by metallic nanowires with excellent electrical and mechanical properties of graphene. The results obtained the combination of nanomaterials with different properties , will help to extend fundamental understanding of the physical interactions between these and other rather different species . Moreover, these functionalities can be utilized for sensing, imaging, or in diagnostic applications.Supplementary Information


In this paper, a high sensitivity and high stability quartz crystal microbalance (QCM) humidity sensor using polydopamine (PDA) coated cellulose nanocrystal (CNC)/graphene oxide (GO) (PDA@CNC/GO) nanocomposite as sensitive material is demonstrated. The PDA@CNC was prepared by the self-polymerization action on the surface of CNC, and it acted as filler material to form functional nanocomposite with GO. The material characteristics of PDA@CNC, CNC/GO and PDA@CNC/GO were analyzed by transmission electron microscope (TEM) and Fourier transform infrared spectroscopy (FTIR), respectively. The experimental results show that the introduction of PDA@CNC into GO film not only effectively enhanced the sensitivity of GO-based nanocomposite-coated QCM sensor but also significantly maintained high stability in the entire humidity range. The PDA@CNC/GO30-coated QCM humidity sensor exhibited a superior response sensitivity up to 54.66 Hz/% relative humidity (RH), while the change rate of dynamic resistance of the sensor in the humidity range of 11.3–97.3% RH is only 14% that is much smaller than that of CNC/GO-coated QCM. Besides, the effect of the PDA@CNC content on the sensitivity and stability of GO-based nanocomposite-coated QCM humidity was also studied. Moreover, other performances of PDA@CNC/GO-coated QCM humidity sensor, including humidity hysteresis, fast response and recovery and long-term stability, were systematically investigated. This work suggests that PDA@CNC/GO nanocomposite is a promising candidate material for realizing high sensitivity and high stability QCM humidity sensor in the entire humidity detection range. Btion (1) . Here, fAs is known, the sensitivity is a key parameter to evaluate the performance of QCM humidity sensors. In the past two or three decades, many researchers have made a lot of efforts to improve the sensitivity of QCM humidity sensors, especially in the development of humidity sensitive materials. Various kinds of materials including ceramics , organicIn our previous works, we reported that graphene oxide (GO), an important derivative of two-dimensional graphene, was a good candidate material for realizing high performance QCM humidity sensors . The strIn this study, we demonstrated a high performance QCM humidity sensor based on PDA@CNC/GO composite. PDA@CNC was chemically synthesized and used as filling material in GO films to form PDA@CNC/GO nanocomposite. PDA@CNC/GO composite, acting as the sensing film, was deposited on the electrode of QCM. Humidity sensing properties of PDA@CNC/GO-coated QCM sensor, including response sensitivity, humidity hysteresis, dynamic property and stability, were systematically investigated. Moreover, the sensitivity and frequency stability of the QCM sensors based on PDA@CNC/GO, CNC/GO and GO materials were compared. Finally, the sensitive mechanism of PDA@CNC/GO-coated QCM sensor was analyzed.GO was synthesized by a modified Hummers method . The obtFirst, 10 MHz AT-cut QCMs with 8-mm crystal diameter and 5-mm electrode diameter were fabricated by Wuhan Hitrusty Electronics, Wuhan, China. The circular electrode of QCM was achieved by thermal evaporation process. A 15-nm/100-nm-thick Cr/Au layer was sequentially coated on 166-μm-thick quartz crystal plate. Prior to sensitive film deposition, the QCMs were cleaned using deionized water and ethanol, respectively. The QCM humidity sensors were fabricated by depositing an equal volume (2 μL) of suspension onto the electrode of each QCM using a micropipette. After that, these QCM sensors were placed in a drying oven and dried at 60 °C for 6 h for sufficient water evaporation. 2, Mg (NO3)2, NaCl, KCl and K2SO4 solutions at 25 °C were used to generate 11.3%, 32.8%, 54.3%, 75.3%, 84.3% and 97.3% humidity levels, respectively. A commercial phase-locked loop oscillator  was used to drive the QCM sensor to produce resonance. This oscillator can provide the frequency and dynamic resistance of QCM. Here, the dynamic resistance of QCM, which is inversely proportional to quality factor (QF), is an important parameter to evaluate the stability of QCM [−1 for 64 scans, WI, USA).The homemade humidity sensing measurement platform is shown in y of QCM . For ins−1 is attributed to the presence of –OH groups. There is a peak at 2910 cm−1 in the spectra of CNC and its composite resulting from the hydroxyl stretching. The two peaks at 1730 and 1640 cm−1 are associated with the C=O stretching and the O–H bending vibration of absorbed water. The peak appearing at 1060 cm−1 is due to the C−O−C pyranose ring skeletal vibrations. The FTIR analysis result confirms that the attachment of CNC and PDA@CNC to GO can be achieved through hydrogen bonding.The material characteristics of the as-synthesized GO, CNC/GO30 and PDA@CNC/GO30 were analyzed by TEM, FTIR and XRD, respectively. f) to the humidity changes (ΔRH) to define the sensitivity of QCM humidity sensor. When humidity changes from 11.3% to 97.3% RH, the frequency shifts of PDA@CNC/GO30-, CNC/GO30- and GO-coated QCM humidity sensors were 4701, 3189 and 2279 Hz, and their corresponding sensitives are 54.66, 37.08 and 26.5 Hz/% RH, respectively. This result clearly indicates that the introduction of PDA@CNC or CNC into GO material can enhance humidity sensitivity since the filler materials offer more adsorption active sites.The humidity sensing measurements of the QCM sensors were performed by exposing the sensors to various saturated salt solutions step by step. First, we compared the humidity sensing response and stability of the QCM humidity sensors coated with the three different sensitive films . Besides the response sensitivity, dynamic resistance of the three sensors were also recorded as a function of humidity to get the evaluation of the stability , and theHR) in Equation (2).ef is the maximum difference in frequency shifts between the adsorption and desorption process and fullf is the full-scale frequency shift of the sensor. It can be calculated that the HR values of PDA@CNC/GO30-, CNC/GO30- and GO-coated QCM humidity sensors are 4.3%, 4.8% and 3.8%, respectively. All three kinds of GO nanocomposite-based QCM sensors exhibit relatively low humidity hysteresis, which is beneficial for the reliability of the humidity sensor in practical application. The observed small HR values is attributed to that the adsorption mechanism of GO-based nanocomposite is weak physical adsorption, which is conducive to water molecule adsorption or desorption of water molecules from sensitive films.Further, the other performances of PDA@CNC/GO30-coated QCM, such as humidity hysteresis, long-term stability and dynamic response, were experimentally studied. The long-term stability is also crucial for the practical application of QCM humidity sensor. We measured the frequency shifts of PDA@CNC/GO30-coated QCM humidity sensor at three given humidity points every three days for three weeks, and the result is shown in The present work demonstrates a high performance QCM humidity sensor using PDA@CNC/GO nanocomposite as sensitive material. Series of experiments were carried out to examine humidity sensing performance of the proposed sensor, including sensitivity, stability, humidity hysteresis, dynamic response and long-term stability. The sensitivity of PDA@CNC/GO30-coated QCM humidity sensor was about two times larger than that of GO-coated QCM sensor. More interestingly, we observed that the change rate of dynamic resistance of PDA@CNC/GO30-coated QCM humidity sensor is only 14%, which was much smaller than that of CNC/GO30- and GO-coated QCM sensors. The results evidently show that the introduction of PDA@CNC into GO films can significantly improve the sensor’s sensitivity while maintaining high stability in the entire humidity range. The explanation of the above experimental results is that the introduction of PDA@CNC into GO films not only provided more hydrophilic groups for water adsorption but also inhibited the swelling of GO-based nanocomposite after water adsorption. Furthermore, the effect of the PDA@CNC content on the performance of GO nanocomposite-coated QCM humidity was also considered. In addition, PDA@CNC/GO30-coated QCM humidity sensor possessed an acceptable humidity hysteresis (4.3% RH), fast response and recovery (37/5 s at 48–97.3% RH) and prominent long-term stability. Our work suggests that PDA@CNC/GO nanocomposite is a promising candidate material for realizing high sensitivity and stability QCM humidity sensors in the entire humidity detection range.


Anorexia, body wasting, inflammation, muscle, and adipose tissue loss are hallmarks of cancer cachexia, a syndrome that affects the majority of cancer patients, impairing their ability to endure chemotherapeutic therapies and reducing their lifespan. In the last 10 years, alterations of protein turnover and impairment of adult myogenesis have been proposed as major contributing factors.Muscle stem cells, including satellite cells, mesoangioblasts, and fibroadipogenic progenitors, were isolated and characterized from C26 colon carcinoma‐bearing (C26) mice. Circulating levels of interleukin‐4/13 (IL4/IL13) were analysed by ELISA, and the effects of IL4 on muscle mass and function, protein synthesis, muscle regeneration, and myogenic progenitor cell number were analysed at both functional (treadmill and grip test) and molecular levels . The Kaplan–Meier test was used to analyse the survival curve of IL4‐treated and IL4‐untreated C26 mice.The administration of IL4 to C26 mice rescued muscle mass by increasing protein synthesis. The IL4 treatment improved performances and prolonged survival of C26 mice. IL4 administration re‐established both number and function of satellite cells and fibroadipogenic progenitors without affecting mesoangioblasts in C26 mice, rescuing myogenesis. Upon IL4 treatment, a high number of cytotoxic lymphocytes and type II macrophages were observed with a subsequent increase in necrotic areas of C26 tumours.The results here presented shed new light on IL4 signalling during muscle wasting and early stages of muscle regeneration that explain the beneficial effect observed in IL4‐treated C26 mice. These findings might aid to develop therapeutic approaches to improve mobility and quality of life in cachectic patients. In this regard, in the muscle of tumour‐bearing animals, satellite cells (SCs) increase in number and accumulate in the interstitial space among the fibres.in vitro.Several studies have clarified that, in addition to SCs, also other cell types can contribute to myogenesis, such as mesoangioblasts (MABs), vessel‐associated muscle progenitors, and fibroadipogenic progenitors (FAPs). Consistently, recent data from our group have demonstrated that BMP‐SMAD signalling blockade improves MAB myogenic differentiation.The aim of the present study is to investigate the mechanisms underlying the impaired myogenesis in cancer hosts, focusing on the role that interstitial cells and cytokines involved in muscle differentiation might play in contributing to cancer‐induced muscle wasting.2, 5% O2, and the medium was refreshed every 2 days. Tumour‐bearing mice were inoculated subcutaneously dorsally with 5 × 105 C26 carcinoma cells. The number of mice per group depends on the experiments as it is indicated in the legends of the respective assays. Where indicated, 1.3 μg per mouse intraperitoneal IL4  was administered every day, starting from Day 5 to the day of sacrifice.liquid nitrogen, and stored at −80°C. Peritoneal lavage was performed with 50 μM of EDTA in phosphate‐buffered saline (PBS) to extract lymphocytes and macrophages. Tumour tissue was digested for 90′ in 1 mg/mL collagenase II and IV 1:1.All animal procedureset al.Four‐month‐old alpha‐sarcoglycan (αSG)‐null mice from the internal colony of Sampaolesi laboratory were generated by the group of K.P. Campbell .Mice were followed until the KU Leuven predetermined humane endpoint criteria were fulfilled. Mice were ultimately sacrificed when C26 cell inoculation determined body mass loss above 20%, combined with an overall health status of severe sufferance, food intake decrease, abnormal appearance and posture , unnatural behaviour , and body temperature below 28°C. During the experiment, mice were separated to avoid self‐mutilation.Mice were introduced to the treadmill belt to test the conditions before the actual test . Later on, the motor speed was set to 10 m/min, with a 1 m/min increase and an uphill inclination of 10°, till exhaustion and >10 s stop.Naja mossambica mossambica, Sigma‐Aldrich) in the tibialis anterior, the gastrocnemius, and the quadriceps of one hind limb. The contralateral muscles were injected with the same volume of PBS as a control. Ten days after the in vivo regeneration experiments, the mice were sacrificed by cervical dislocation, and muscles were processed for histological analysis. For the experiment of muscle injury in parallel to the survival experiment , mice were sacrificed when all the signs of severe sufferance were reached and body temperature was below 28°C.Mice received 50 μL injection of 10 μM solution of cardiotoxin  and euthanized 30 min after injection.2, 5% O2, and the medium was refreshed every 2 days. Myoblast differentiation was obtained with the same medium as for C2C12 differentiation. After tissue explant, adult Mesoangioblasts (MABs) were sorted for ALP+ and induced to express GFP  were performed in αSG‐null mice (n = 5 for each group). During the entire treatment, mice were maintained with cyclosporine ; 21 days post‐injection mice were sacrificed and compared with sham‐operated mice .Adult SCs were isolated by enzymatic digestion with 0.01% collagenase D and 0.06% pancreatine during 90′ digestion of minced murine muscle and pre‐plated on 0.1% gelatin‐coated dishes to remove fibroblasts. Cells were counted and plated on 0.1% collagen for experiments of cell proliferation in high‐glucose DMEM supplemented with 20% FBS, 1 mg/mL of sodium pyruvate, 1 IU/mL of penicillin and streptomycin, and 1 mg/mL of non‐essential amino acids, 50 μg/mL of gentamicin, and 1:100 chick embryo extract . The plates were incubated at 37°C, 5% COP as for . After s2 in air‐humidified chamber in high‐glucose DMEM with glutamine, supplemented with 10% FBS, 1 mg/mL of sodium pyruvate, and 1% penicillin/streptomycin (Thermo Fisher Scientific). When the cells reached confluency, growth medium was replaced with differentiation medium, supplementing high‐glucose DMEM with 2% horse serum, 1 mg/mL of sodium pyruvate, and 1 IU/mL of penicillin/streptomycin. The plates were incubated at 37°C, 5% CO2, 5% O2, and the medium was refreshed every 2 days.C2C12 myoblast cells were maintained at 37°C in 5% COIL4Ra and IL13Ra, endoribonuclease‐prepared siRNAs against IL4Ra and IL13Ra  were applied following the manufacturer's instructions. Briefly, C2C12 at Day 2 of myotube differentiation was transfected using Lipofectamine 2000 and Opti‐MEM. At different time points after transfection , cells were lysed for RNA extraction or WB analysis. RNA Mini Kit was used for RNA isolation, and genomic DNA traces were removed with Turbo DNase, following the manufacturer's instructions; 500 μg of RNA were reverse‐transcribed with the SuperScript III kit. The resulting cDNA was transferred in a 384‐well plate pre‐filled with 250 nM primers. Platinum SYBER green mix was added to a final volume of 10 μL per well. The RT–qPCR was performed for 40 cycles  and read on a ViiA 7 qPCR plate reader. The list of murine primers used is available in TableIn order to efficiently knockdown TableStaining was performed with primary antibody Table during 3IL4 and IL13 serum levels were detected by a commercially available mouse ABTS‐ELISA kit, used according to the manufacturer's instructions (Peprotech). Serum from each animal (50 μL) was assayed in triplicate. Quantitative calibration was obtained performing a standard curve with recombinant mouse IL4 and IL13.TableTissues (7 μm muscle tissue and 5 μm tumour tissue) and cells were fixed with 4% paraformaldehyde (Polysciences Europe GmbH) in PBS and permeabilized with 0.2% Triton X‐100 in PBS containing 1% (w/v) bovine serum albumin (BSA). A blocking solution containing donkey serum  was applied, and primary antibodies  supplemented with 10 mM sodium fluoride, 0.5 mM sodium orthovanadate, 1:100 protease inhibitor cocktail (Sigma‐Aldrich), and 1 mM phenylmethylsulfonyl fluoride. Equal amounts of protein (40 μg) were heat‐denatured in sample‐loading buffer , resolved by SDS‐polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes . The filters were blocked with Tris‐buffered saline containing 0.05% Tween and 5% non‐fat dry milk (Sigma‐Aldrich) and then incubated overnight with the indicated dilutions of different primary antibodies .All results were expressed as mean ± standard deviation, with the exception of gene expression (mean ± SEM). Representative WBs were selected from independent experiments. When data distribution approximated normality and two groups were compared, a Student's Figuretibialis anterior, gastrocnemius, and quadriceps . The expression of terminally differentiated myotube markers let us conclude that ex vivo C26‐SCs behave comparably with C‐SCs.The number of SCs isolated from muscles of C26 hosts (hereafter C26‐SCs) was higher than that obtained from C muscles . In order to test if C‐MABs and C26‐MABs display the same ability in vivo, these cells were injected intramuscularly into αSG‐null mice.+ fibres expressing αSG, clearly originated from the fusion of the injected C‐MABs and C26‐MABs ‐positive cells FigureF were isFigureFigurein vitro.Finally, the majority of interstitial muscle cells, isolated from the muscle of C26‐bearing mice and put in culture, differentiated to adipocyte‐like cells, a pattern that was not observed in cultures prepared with cells isolated from C muscles. Increased PLIN1 expression of Oil‐Red‐O‐positive cells . The low availability of efficient antibodies for WB let however any conclusion on the expression of these molecules still at a speculative level. Finally, while circulating IL4 was comparable in both C26 hosts and C mice . IL4 treatment improved C26‐induced muscle wasting in terms of both muscle mass and cross‐sectional area (CSA) stabilizing this latter to the level of C , while it remained comparable with Day 0 in tumour‐bearing mice receiving IL4  and type II macrophages  was higher in tumours from IL4‐treated than IL4‐untreated C26 hosts  in IL4‐treated hosts.IL4 treatment could contribute to stimulate the immune system against tumour cells. To investigate this point, C26 tumours from both IL4‐treated (Day 31 after tumour transplantation) and IL4‐untreated (Day 13 after tumour transplantation) animals were analysed in terms of lymphocyte and macrophage content. The results showed that the percentage of cytotoxic lymphocytes , which could be involved in tumour cytotoxicity and necrosis. These results suggest that IL4 administration to C26‐bearing mice, far from stimulating cancer growth, increases the immune response against the tumour. However, this observation is in apparent contrast with the well‐known anti‐inflammatory action of IL4. In this regard, further experiments are required to clarify if systemic IL4 administration might exert different actions at the tumour and muscle levels, respectively. Consistently, the observations here reported show that in vitro IL4 is able to modulate myogenesis but not to affect C26 tumour cell growth curve, suggesting that while the effects on muscle are directly mediated by IL4, those on the tumour could depend on additional mediators.A possible limitation in the present study is that increased tumour mass occurs when IL4 is administered to C26‐bearing mice. In this regard, in colorectal cancer stem cells, protection from apoptosis is achieved by autocrine production of IL4 through up‐regulation of anti‐apoptotic mediators.In conclusion, these results demonstrate the importance of IL4 as a cytokine able to impinge on the inflammatory system, promoting an anti‐inflammatory muscle micro‐environment, and to raise the state of activation of anabolic kinases involved in the maintenance of muscle protein synthesis. Of interest, IL4 shares at least some beneficial actions with exercise, previously shown to reduce systemic inflammationD.C. participated in conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; R.D. participated in flow cytometry collection and assembly of data, data analysis and interpretation; F.P. participated in data interpretation; D.N. participated in data interpretation; P.C. participated in conception and design, data analysis and interpretation, manuscript writing, and final approval of the manuscript; M.S. participated in conception and design, data analysis and interpretation, manuscript writing, and final approval of the manuscript.None declared.+ C‐ and c) GFP+ C26‐MABs in co‐culture with C2C12 at day 0 and 5 of myotube differentiation. d) qRT‐PCR for the expression of IL4, IL4Ra1, IL13 and IL13R in C and C26 muscles normalized for the housekeeping genes Gapdh, HPRT, TBP. Significance of the differences: *p < 0.05, ****p < 0.0001 vs C.Figure S1: Characterization of C‐ and C26‐SCs and MABs. a) IF of SCs from C and C26 muscles stained for ACTN2 (red) and MyoD . Nuclei were stained with HOECHST (blue). Scale bar: 500 μm. WB for MyHC and aTub on b) GFPin vitro with 10, 100 and 500 ng/ ml IL4. b) Absorption curve of IL4 in Balb/c mice at different time points after 1.3 ug IL4 administration (n = 3 per time point). c) Tumor weight in C26 and C26 + IL4 at the day of the sacrifice . d) Quadriceps muscle weight of C, IL4, C26 and C26 + IL4 mice (C: 410 ± 33 mg/100 g i. b. w.). Representative flow cytometry analysis on cells extracted after peritoneal lavage from  1 mg/kg LPS injected mice (as positive control), or C26 and C26 + IL4 peritoneal lavage was analyzed for the main markers of (f) lymphocytes and (g) quantified or for (i) macrophage populations and (j) quantified. Significance of the differences **p < 0.01, ***p < 0.001, ****p < 0.0001 vs C; ++p < 0.01, +++p < 0.001 vs IL4; $p < 0.05, $$$p < 0.001 vs C26.Figure S2: IL4 treatment. a) C26 number of cells treated for 72 h Figure S3: Flow cytometry analysis of circulating immune cells. Representative flow cytometry analysis of the circulating immune cells from C26 and C26 + IL4 mice for the main markers and quantifications of (a) lymphocytes and (b) macrophages. Significance of the differences $$$p < 0.001 vs C26.Figure S4: analysis of other important protein expressions in muscle tissue. a) WB for p38 MAP‐Kinase and (b) quantification. c) WB for nuclear extract of P‐STAT3 and P‐STAT6 with  respective quantifications. f) WB for Atrogin1 and TRIM32 with  respective quantification. i) WB for Beclin1, LC3B, p62 and (j ‐ l) respective quantifications. m) WB for the comparison of p62 levels between C26 and C26 + IL4 and (n) quantification. All the values were normalized for the total protein levels and on aTub or GAPDH expression levels. Significance of the differences is reported as *p < 0.05, **p < 0.01, ***p < 0.001 vs C; ++p < 0.01, +++p < 0.001 vs IL4; $p < 0.05 vs C26.Figure S5: Numbers of cells extracted from muscle of C, IL4, C26 and C26 + IL4 normalized for the muscle mass. a) Cells were counted immediately after tissue digestion. b) IF on muscle slides for the expression of PDGFRa (green) and Ki67 (magenta) from C, IL4, C26, C26 + IL4 muscles. HOECHST (blue) was used to stain nuclei. The arrows indicate double positive nuclei surrounded by a green signal. *p < 0.05 vs C; ++p < 0.01 vs IL4; $$$ p < 0.001 vs C26. Scale bar: 100 μm.Figure S6: IL4‐treated muscles do not show impairment in regeneration after CTX injury. a) Average of fibre CSA in muscles after 10 days of CTX injection. b) H&E staining of muscle sections taken at longer time points after CTX injury was induced. In particular, for CTX‐C26 (n = 5), 9 to 12 days after CTX injury, and for CTX‐C26 + IL4 mice (n = 5), 16 to 30 days after CTX injury. Significance of the differences ***p < 0.001 vs C; +++p < 0.001 vs IL4; $$$p < 0.001 vs C26.Figure S7: Increasing concentrations of IL4 dos not rescue 50 ng/ ml TNFa‐impaired myotube differentiation. a) qRT‐PCR for the expression of IL4, IL4Ra, IL13 and IL13R in C2C12 at day 0 and 5 of myotube differentiation normalized for the housekeeping genes Gapdh, HPRT, TBP. b) C2C12 myoblasts at day 2 of differentiation were analyzed for IL4R expression on the levels of aTUB. c) MyHC levels by WB were normalized for aTub and quantified in C2C12 treated with low concentration of TNFa (50 ng/ml) and (d) 100 ng/ ml IL4 or (e‐f) 200 ng/ml IL4. g) Silencing for IL4R1a and (h) for IL13R1a in myotubes was measured by qRT‐PCR during the three last days of differentiation . i) Levels of Myomixer (Mymx), Myomaker (Mymk) and Myomerger (Gm7325s and Gm7325l) were analyzed on the IL4R1a silenced cells by qRT‐PCR normalized for the housekeeping genes Gapdh, HPRT, TBP. j) WB and (k) quantification for ESGP (Myomerger) on the levels of GAPDH in CTX‐C CTX‐IL4, CTX‐C26 and CTX‐C26 + IL4 muscle protein extract. Significance of the differences *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs C; +p < 0.05, +++p < 0.001 vs IL4. £p < 0.05 vs CTX‐C; ^p < 0.05 vs CTX‐IL4.Click here for additional data file.


We use scanning probe photocurrent imaging to resolve nanoscale variations of the Seebeck coefficient occurring at these domain walls. The observed features become enhanced in a range of mid-infrared frequencies where the hexagonal boron nitride substrate is optically hyperbolic. Our results illustrate the capabilities of the nano-photocurrent technique for probing nanoscale electronic inhomogeneities in two-dimensional materials.Quasi-periodic moiré patterns and their effect on electronic properties of twisted bilayer graphene have been intensely studied. At small twist angle  Here, the authors use scanning probe photocurrent imaging to resolve nanoscale variations of the Seebeck coefficient occurring at domain walls separating micron-scale AB and BA stacking regions in twisted bilayer graphene, and observe hyperbolic enhancement of the photocurrent pattern. A key feature of TBG is the moiré superlattice: a long-range variation in the atomic stacking arising from geometric interference of the lattice periodicities in the two graphene sheets. Scanning probe studies of TBG with θ ∼ 1.1° demonstrated spatial variations in the electronic properties occurring on the length scale of tens of nanometers9.Twisted bilayer graphene (TBG), consisting of two graphene sheets rotated with respect to each other, has emerged as a tunable platform for studying exotic electronic phases. Transport experiments have revealed that when the graphene layers are twisted by a magic angle of θ ≈ 0.01° and prone to atomic relaxation. In the relaxed state, the Bernal stacked domains (AB and BA) dominate while the less stable stacking configurations are reduced to a network of narrow domain walls (DWs). Transmission electron microscopy (TEM) measurements have shown that the DWs are 6–9 nm wide10. Previous transport11, nano-infrared13, and scanning tunneling microscopy (STM)14 studies have revealed the existence of topological states at the DWs when an electronic bandgap is opened by a sufficiently large interlayer bias between the graphene sheets. At smaller interlayer biases, the change in the atomic stacking across the DW still leads to a change in the electronic properties.In minimally twisted bilayer graphene (MTBG), the moiré pattern periodicity is large, e.g., 140 nm for 15. Previous nano-photocurrent experiments have resolved charge inhomogeneities and grain boundaries in monolayer graphene15 and mapped variations in twist angle of TBG at twist angles θ > 1°16. Here, we use scanning nano-photocurrent imaging to study domain walls in MTBG. We show that the photocurrent patterns arise from DC Seebeck coefficient variations occurring at the DWs on a nanometer length scale. We further propose and demonstrate a mechanism that utilizes the intrinsic hyperbolicity of the hBN substrate to enhance the DW features in photocurrent images.Scanning nano-photocurrent imaging has emerged as a novel optoelectronic probe capable of resolving changes in DC transport properties of graphene with nanometer-scale spatial resolution19. Photocurrent images are acquired by raster scanning the tip across the sample. Our technique overcomes the diffraction limit and provides a spatial resolution of about 20 nm while also allowing for simultaneous nano-infrared imaging15. Our device consists of two graphene layers with a minimal relative twist encapsulated between 37 nm bottom hexagonal boron nitride (hBN) layer and 6 nm top hBN layer. The entire stack rests on a 285 nm SiO2/Si substrate with the SiO2 layer serving as the gate dielectric. Piezoresponse force microscopy (PFM)20 before encapsulation of the device revealed domain walls with a periodicity of about 500 nm ω = 900 cm−1. We use a color scheme that enables easy identification of the sign of the photocurrent: red and blue represent positive and negative currents respectively while white represents regions where the measured current is zero, thus highlighting the zero-crossing contours. Some of the zero-crossing contours form easily identifiable lines in the y-direction while others form a meandering pattern. On closer inspection, we find a series of fine structures in the photocurrent image that form a hexagonal lattice. These features are more clearly revealed in the map of the photocurrent gradient, dIPC/dx, shown in Fig. IPC/dx image, the vertical domain walls appear to be more intense because of the contact configuration used in our experiments, as explained in Supplementary Note Figure VG = +4V . In this mechanism, the absorption of incident light generates hot carriers in graphene. When the hot carriers encounter variations in the Seebeck coefficient, a thermoelectric voltage is generated, which drives a current through the sample. The spatial profile of the measured current is, therefore, directly related to the Seebeck coefficient profile in the sample. PTE shows several characteristic features in experiments. First, since Seebeck coefficient is antisymmetric with respect to the sign of the carriers, the resulting photocurrent patterns also change sign when the carrier type changes from holes to electrons21. Second, the Seebeck coefficient of bilayer graphene rapidly diminishes as the carrier density increases23. Therefore, any variations in the Seebeck coefficient and the resulting photocurrent must also become small. Both features are present in our data, strongly suggesting that the photocurrent patterns we observe arise from PTE.Previous theoretical1 a “lightning-rod model” in which the tip is represented by a conducting hyperboloid and2 a simplified common approximation of the tip by a vertically oriented point dipole  will produce a pattern similar to the experiment, as we demonstrate in Supplementary Note While the first-principles Seebeck coefficient profile produced a photocurrent pattern similar to the experiment, we note that our experiment is not sensitive to the fine details of the Seebeck coefficient at the domain wall. In fact, any change in Seebeck on a length scale significantly shorter than the spatial extent of the hot carriers , where hBN transverse dielectric constant in the xy-plane becomes negative  could reveal such features in future photocurrent experiments The properties of bilayer graphene depend not only on the carrier density but also on the interlayer bias. In our experiment, we have a single Si back gate, which allows us to control the carrier density accurately. Here, we describe our estimate of the interlayer bias values for different gate voltages.36. For a given gate voltage, we can directly calculate the displacement field below the graphene layers:∈lower and dlower are the dielectric constant and thickness of the SiO2 dielectric layer. As we have no top gate, the displacement field above the graphene layers First, we assume that the interlayer bias is zero at charge neutrality point 37 to estimate the interlayer bias Vi from EF, we keep Vi fixed and vary the Fermi energy EF until the carrier density we calculate with a tight-binding model matches the value expected from capacitance calculations. Supplementary Fig. EF and Vi for several gate voltages. We find that the estimated Fermi energy is linear with gate voltage. At small displacement fields, the band structure of bilayer graphene can be well approximated to be parabolic38. In two dimensions, a parabolic dispersion leads to a constant density of states and a linear dependence of the Fermi energy on carrier density, which is consistent with our estimate. We note that the carrier densities considered in this manuscript (Figs. 39.We use ref. entclass1pt{minimaentclass1pt{minima40 at T = 200 K. For the ω = 900 cm−1 experiments, we used a CO2 laser and for the Reststrahlen band experiments, we used a tunable quantum cascade laser from Daylight Solutions. The incident laser power was around 20 mW in all cases. The current was measured using a Femto DHPCA-100 current amplifier. To isolate the photocurrent contributions from the near-fields localized under the tip, the measured current was demodulated at a harmonic n of the tapping frequency. In this work, we used n = 3 for room temperature experiments and n = 2 for low-temperature experiments.Room temperature nano-photocurrent measurements were performed in a commercial s-SNOM from Neaspec GmbH. Low-temperature nano-photocurrent measurements were performed in a home-built SNOM within an ultra-high vacuum chamberSupplementary Information


The International Safe Communities movement was established in 1986, and since that time has developed into an international collaborative network comprised of the WHO CCCSP/ ISCCC, regional and national safe community organizations, support centers and communities. Purpose to review the outcomes of communities who have achieved designation as an international safe community over the 32-year period from 1986 to 2018.An audit of the ISCCC database and website was undertaken. Any inconsistencies were checked for accuracy against the original designation application.Since the safe community movement began in 1998 four hundred and three International Safe Communities have been designated. Data was available for analysis regarding 361 communities. Thirty-eight communities, all designated prior to 2006, had been removed from the data base prior to this data being handed over to the ISCCC. The data set is complete from 2006 onwards. The program has a high reach, 86 million people have been served by a safe community program that has achieved International Safe Community Designation on at least one occasion (1.1% of the world population). Forty-nine millions of these are served by safe community programs whose certification remains active. In 2018 there were 39 certifications (13 designations and 26 redesignations), covering a population of 7.1 million people. One hundred and sixty-one (40%) of the original 403 communities have maintained a safe community certification. Two hundred and forty-four (68%) communities only sought initial designation, 92 (25%) were designated twice, 22 (6%) were designated three times and 3 (1%) were designated four times. International Safe Communities vary considerably in size from the smallest with a population of 1,000 inhabitants to the largest of 12 million. However, the distribution is skewed towards smaller communities (5th percentile = 6,000, a median of 90,000, a mean of 240,000 and a 95th percentile of 700,000. The International Safe Community movement has accomplished remarkable growth in the 30 years since it began in Sweden in the late 1980s. The program has achieved high reach. Four hundred and three communities have been designated by the end of 2018 with a population footprint of 86 million people.International Safe Communities, Designation, Sustainability, Evaluation