Saccharomyces cerevisiae, N-acetyl glutamate kinase (NAGK), which catalyzes the phosphorylation of N-acetyl glutamate to form N-acetyl glutamyl-5-phosphate, is one of the rate-limiting enzymes in the ornithine and arginine biosynthetic pathways. NAGK activity is strictly regulated via feedback inhibition by the end product, arginine. We previously reported that the Thr340Ile variant of NAGK was insensitive to arginine feedback inhibition and that the interaction between Lys336 and Thr340 in NAGK may be important for arginine recognition. In the present study, we demonstrated that amino acid changes of Thr340 to Ala, Leu, Arg, Glu, Ile, and Asn removed arginine feedback inhibition, although the Thr340Ser variant was subject to the feedback inhibition. Therefore, these results indicate that the arginine-binding cavity formed via the interaction between the carbonyl group in the main chain of Lys336 and the hydroxyl group in the side chain of the residue at position 340 is critical for arginine recognition of NAGK. In addition, we newly identified two mutations in the ARG5,6 gene encoding the Cys119Tyr or Val267Ala variant of NAGK of sake yeast mutants with intracellular ornithine accumulation. Although it is unlikely that Cys119 and Val267 are directly involved in arginine recognition, we found here that two variants of NAGK were insensitive to arginine feedback inhibition and contributed to high-level production of ornithine. Structural analysis of NAGK suggests that these two amino acid substitutions influence the sensitivity to Arg feedback inhibition through alterations in local conformation around each residue.In the yeast IMPORTANCE Ornithine has a number of physiological benefits in humans. Thus, an Orn-rich alcoholic beverage is expected to relieve feelings of fatigue after drinking. In the yeast Saccharomyces cerevisiae, N-acetyl glutamate kinase (NAGK) encoded by the ARG5,6 gene catalyzes the second step in ornithine and arginine biosynthesis, and its activity is subjected to feedback inhibition by arginine. Here, we revealed a role of key residues in the formation of the arginine-binding cavity which is critical for arginine recognition of NAGK. In addition, we analyzed novel arginine feedback inhibition-insensitive variants of NAGK in sake yeast mutants with ornithine overproduction and proposed that the amino acid substitutions in the NAGK variants destabilize the arginine-binding cavity, leading to the lower sensitivity to arginine feedback inhibition of NAGK activity. These findings provide new insight into the allosteric regulation of NAGK activity and will help to construct superior industrial yeast strains for high-level production of ornithine. Ornithine (Orn), a nonproteinogenic amino acid, functions in the urea cycle, which detoxifies ammonia generated by the degradation of amino acids in protein to urea. Various organisms and foods contain large amounts of Orn –3. In hu–Saccharomyces cerevisiae synthesizes Orn from glutamate (Glu) via intermediates such as N-acetyl l-glutamate (NAG) and N-acetyl glutamyl-5-phosphate, N-acetyl glutamyl-γ-semialdehyde, and N-acetyl Orn in mitochondria  and the other being N-acetyl glutamyl-5-phosphate reductase (Arg5), between residues 510 and 540 , which is encoded by the ARG2 gene, is another rate-limiting step of Arg biosynthesis. NAGS transfers the acetyl group to Glu to produce NAG, and the protein or expression level of this enzyme is tightly regulated due to the Arg feedback inhibition  from Glu. γ-Glutamyl kinase (GK) encoded by the PRO1 gene is the rate-limiting enzyme of Pro biosynthesis from Glu, and its activity is strictly regulated by Pro feedback inhibition SaccharomS. cerevisiae . Furthermore, our structural analysis of the Thr340Ile variant of NAGK predicted that the Arg-binding cavity consists of Lys265, Ser285, Lys336, Glu337, Thr340, and Gly345 and that this cavity may be stabilized through the interaction between the hydroxyl group in the side chain of Thr340 and the carbonyl group in the main chain of Lys336  that were resistant to the Pro toxic analogue azetidine-2-carboxylate (AZC) derived from a diploid sake yeast strain of revisiae . In addif Lys336 .ARG5,6 gene encoding the Cys119Tyr and Val267Ala variants of NAGK, respectively, leading to an increase in intracellular Orn. Several genetically modified microorganisms, such as Corynebacterium crenatum  that was resistant to AZC by conventional mutagenesis. Strains A902-6 and A902-8-3 each had a novel mutation in the crenatum  and S. crevisiae , have beIn the present study, using traditional chemical mutagenesis, we isolated a new Pro- and Orn-accumulating strain, A902-8-3, from AZC-resistant mutants of diploid sake yeast strain K901 in addition to previously isolated strains A902-4 and A902-6 . As showARG5,6 gene encoding the Thr340Ile variant of NAGK. The NAGK activity of this variant was insensitive to feedback inhibition by Arg, leading to overproduction of Orn . As expected, the ARG5,6 gene sequence in A902-6 contained a homozygous mutation, which was a single base change at position 356, leading to one amino acid change of Cys to Tyr at position 119. On the other hand, A902-8-3 had a heterozygous mutation, i.e., a single base change at position 800, which led to one amino acid change of Val to Ala at position 267. In contrast, strains A902-6 and A902-8-3 harbored no other mutations in the genes encoding the rate-limiting steps for Orn (ARG2) and Pro (PRO1) biosynthesis  and mutated ARG5,6 genes . In addition, the importance of Thr340 in the mechanism underlying the feedback inhibition of NAGK was still unknown. Therefore, we also designed a series of mutated ARG5,6 genes  that replaced Thr340 with various amino acids  that have a side chain with a similar or different size, charge, and polarity.To analyze the arg5,6Δ expressing the wild-type and mutated ARG5,6 genes  or higher than (A902-6 and A902-8-3) that in the parent strain, K901  cells, purified the recombinant wild type (WT) and variants  of NAGK tagged with 6×His at the amino terminus from E. coli cell extracts , and measured enzyme activities of NAGKs under conditions without or with Arg. As shown in We previously showed that the mutated activity . We ther50Arg) of WT and T340S were less than 0.2 mM. In contrast, the newly identified variants (C119Y and V267A) exhibited higher IC50Arg  than that of WT, suggesting that Cys119 and Val267 are important for the regulation of NAGK activity. These results indicate that the novel NAGK variants (C119Y and V267A) were less sensitive to feedback inhibition by Arg than WT. Moreover, in the NAGK variants at position 340 , each IC50Arg (ranging from 0.78 mM to 23.98 mM) was higher than those of the WT and T340S enzymes. It should be noted that the IC50Arg of the T340I variant  as a template. The results showed that replacement of Thr340 with Ser can still form the hydrogen bond with Lys336 as well as the WT enzyme because the hydroxyl group in the side chain of Ser340 is located near the carbonyl group in the main chain of Lys336 (2.4 Å)  in the side chain was higher than that of the acidic (Glu), the basic (Arg), or the small (Ala) amino acid in the side chain  , resulti (2.4 Å) . In contde chain . These fS. cerevisiae NAGK reported that the binding of inhibitor Arg caused a conformational change with an axis that crosses obliquely the central β sheet (β11-sheet) including Val267-Tyr268, resulting in transition from the closed-active form to the open-inactive form   including Lys336 and Glu337 involved in the interaction with Arg . The steig. S4A) . One posARG5,6 genes encoding the Cys119Tyr and Val267Ala variants of NAGK. These amino acid substitutions weakened the Arg feedback inhibition of NAGK, leading to high-level production of both Pro and Orn in yeast cells. Structural analysis suggests that the two amino acid replacements Cys119Tyr and Val267Ala desensitize Arg feedback inhibition through alterations in local conformation around each residue. These results will contribute to the development of superior industrial yeast strains for high-level production of Orn, which has a number of physiological benefits.In the present study, functional analysis of the Thr340 variants of NAGK revealed that the Arg-binding cavity formed through the interaction between Lys336 and Thr340 is critical for Arg recognition of NAGK. In addition, we newly analyzed diploid sake yeast mutants A906-2 and A902-8-3, which overproduced not only Pro but also Orn. These two strains possessed novel mutations in the S. cerevisiae strain BY4741 (MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0) derived from strain S288C and diploid strain Kyokai no. 901 (K901) in this study. Strain BY4741 arg5,6Δ , which was provided by EUROSCARF , was used for the expression of the WT and mutated ARG5,6 genes. The media used for growth of S. cerevisiae were a nutrient-rich YPD medium , a synthetic dextrose (SD) minimal medium , and SD medium containing 0.04% leucine, 0.008% histidine, and 0.008% methionine (SD+Leu+His+Met). AZC was added as a supplement at a concentration of 1 mg/mL for the screening of Orn-accumulating cells.We used Escherichia coli strains DH5α [F− λ− ϕ80lacZΔM15 Δ(lacZYA argF) U169 deoR recA1 endA1 hsdR17(rK− mK+) supE44 thi-1 gyrA96] and BL21(DE3) [F−ompT hsdS(rB− mB−) gal dcm λ(DE3) (lacI lacUV5-T7 gene1 ind1 sam7 nin5)] were used for construction of expression plasmids and for expression of the recombinant NAGKs, respectively. E. coli strains were cultured in Luria-Bertani (LB) medium  containing 100 μg/mL ampicillin. If necessary, 2% agar was added to solidify the medium.Orn-accumulating mutants were isolated by conventional mutagenesis of the diploid sake strain K901 as described previously .S. cerevisiae cells with a UF-Amino Station  as described previously  and ARG5,6Rv (BamHI)  and was introduced into the HindIII-BamHI site of yeast centromere plasmid pRS416 by the In-Fusion HD cloning kit  to construct pRS416-ARG5,6. In-Fusion products were then digested with DpnI before introduction into E. coli DH5α cells. The point mutations were introduced into the ARG5,6 gene on pRS416-ARG5,6 with mutagenic primers listed in Table S1, leading to the T340A, T340L, T340R, T340E, T340N, T340S, T340I, V267A, and C119Y substitutions on NAGK. PCR products were then digested with Dpnl before introduction into E. coli DH5α cells. Plasmids of pRS416 harboring the wild-type or mutated ARG5,6 gene were further confirmed by DNA sequencing using the BigDye Terminator v3.1 cycle sequencing kit . Strain BY4741 arg5,6Δ was transformed with pRS416 harboring the wild-type or mutated ARG5,6 gene by the lithium acetate method with slight modification , the amino-terminal NAGK (residues 58 to 513), and the carboxyl-terminal N-acetyl glutamyl phosphate reductase (Dpnl before introduction into DH5α cells. Plasmids pQE-2-NAGK-WT and pQE-2-NAGK-variants were further confirmed by DNA sequencing. E. coli strain BL21(DE3) was transformed with pQE-2-NAGK-WT or pQE-2-NAGK-variants.To construct plasmids for expressing the fication . Yeast teductase . The polE. coli BL21(DE3) cells harboring pQE-2-NAGK-WT or pQE-2-NAGK-variants were cultivated in 5 mL of LB medium containing 100 μg/mL ampicillin and grown at 37°C to an optical density at 600 nm (OD600) of 0.6 to 0.8. Protein expression was induced by adding isopropyl-β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and cells were cultured at 18°C for 18 h. Cells were harvested by being placed on ice for 5 min and centrifuged at 4°C for 10 min at 3,000 rpm. The cell pellet was washed with ice-cold sonication buffer , and the intact cells were stored at −80°C if they were not used immediately. For purification of recombinant NAGKs, cell pellets were suspended in ice-cold sonication buffer. The cell mixture was subjected to sonication  by a US-150T ultrasonic generator , was subsequently centrifuged at 15,000 rpm for 20 min at 4°C, and then filtered with an 0.45-μm-diameter filter. Crude protein was loaded by gravity on Ni-Sepharose 6 Fast Flow resin , equilibrated with sonication buffer. The resin was washed in sonication buffer with 20 mM imidazole and then with 70 mM imidazole-containing sonication buffer. Purified proteins were eluted by 500 mM imidazole-containing sonication buffer. Proteins were quantified using Bio-Rad protein assay  and subjected to SDS-polyacrylamide gel electrophoresis .2, 1 mM PEP, 20 U PK, 30 U LDH enzymes from rabbit muscle , 0.25 mM NADH, 100 mM HEPES (pH 7.5), and 5 μg of NAGK in a final volume of 300 μL. The NAGK reaction mixture was prewarmed at 30°C for 1 min without NAG, and then NAG was added to start the NAGK reaction. The rate of the decrease in absorption at 340 nm was measured consecutively. NAGK activity was calculated from the initial linear rate with the extinction coefficient (ε340 nm = 6.22 mM−1 1 cm−1) of NADH. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of NAD+ per min. To determine the 50% inhibitory concentration (IC50) values of NAGK activity, NAGK activity was measured by adding Arg, and kinetic parameters of each enzyme were calculated with GraphPad Prism version 9 (GraphPad Software) using nonlinear regression analysis.NAGK activity was measured by an enzyme-coupled system with lactate dehydrogenase (LDH) and pyruvate kinase (PK) as previously described . BrieflyS. cerevisiae  as the template structure as follows: Thr340 was replaced with Ala, Leu, Arg, Glu, Asn, Ser, and Ile, respectively, or Cys119 and Val267 were changed to Tyr119 and Ala267, respectively, using PyMOL (http://www.pymol.org). Figures of NAGK variant structures were drawn using PyMOL.To analyze the effect of amino acid substitution on NAGK, we predicted the structure of the Arg-binding site in the variant NAGKs using the crystal structure of NAGK bound to Arg from The data underlying this article are available in the article.


Superlattice nanostructures are correspondingly introduced to minimize the thermal conductivity of nanomaterials. Herein we computationally estimate the effect of total length and superlattice period ( However, the semiconductor and transistor applications of graphene are challenging due to its zero bandgap. Furthermore, thermal performance optimization of graphene, for thermoelectric energy conversion and optoelectronic devices, requires chemically-hybridized graphene with some other 2D nanomaterials, such as boron-carbide14, nitrogen-carbide15, graphane16, beryllium-oxide18, silicone-germanium19, or boron-nitride25. The resulting heterostructures are named hybrid superlattice nanosheets26.From the first exfoliation of graphite to graphene synthesis, two-dimensional (2D) nanomaterials have become worth of profound consideration. Graphene, sp3 hybridization that create C–H bonds, is a semiconductor and insulator having a bandgap in the range of 3.5–4.4 eV27. Graphane can be found in different conformations; however, the so-called chair conformation, in which hydrogen atoms alternate their connection below and above the graphene sheet28, is known as the most stable one. Graphane structure was first experimentally reported in 2009 by Elia et al., obtained through graphene exposure to cold hydrogen plasma at a pressure of 0.1 mbar29. Then, properties of pristine or hybrid forms of graphane were reported in the pertinent literature. Namely, the heat capacity of graphane was estimated by 29.32 ± 0.23 J mol−1 K-1, as predicted using molecular dynamics (MD) simulation by Neek-Amal30. The 2D Young's modulus of graphane was computed to be 245 N m−1 employing density functional theory (DFT), smaller than the value of Young's modulus reported for 340 ± 50 N m−1 graphene31.Graphane, i.e., fully hydrogenated graphene possessing carbon atoms in the form of sp32 reported that complete hydrogenation of graphene reduces the thermal conductivity by about 70–80% due to the softening of G-band phonon modes upon the transition of sp2 sp3 bond. Rajabpour et al.33 found the thermal rectification and interface thermal resistance of hybrid graphene-graphane nanoribbons, demonstrating the potential usage of this hybrid nanoribbon as a promising thermal rectifier. The thermal conductivity of multilayer graphene/graphane/graphene heterostructure nanoribbons was also studied using MD simulation by Kim et al.34. They reported that the chemical integration of graphene with graphane creates heteronanosheets where a decrease in the thermal conductivity of graphene to a desirable level by 96% can be obtained. These cases mentioned above indicate the effort of researchers to manipulate the thermal conductivity of such structures towards the fabrication of nanodevices with controlled thermal conductivity. In this regard, superlattice nanostructures are introduced for minimizing thermal conductivity. Such superlattice structure consists of a repeating unit containing different materials with a specified periodic length  was estimated to be minimum (32 nm) for the same superlattice period. It was found that the suppression of coherent phonon thermal transport is achieved by increasing the number of interfaces per unit cell. Correspondingly, the Fibonacci expression is enlarged due to the growth of the hindering phonon coherence along the superlattice nanoribbon36. Wang et al.37 resulted in infinite thermal conductivities of 16.08, 15.95, 5.60 W m−1 K−1 for silicene, pure germanene, and silicene-germanene superlattice nanoribbon, respectively. The dependencies of thermal conductivity on the total length, temperature, and the temperature difference between hot and cold baths for the C3N/C2N superlattice nanosheets were studied by Razzaghi et al. through the MD simulation37. They obtained the values of 23.2 W m−1 K−1, and 24.7 nm for the minimum thermal conductivity and the phonon mean free path, respectively, at a superlattice period of 5.2 nm. Moreover, they reported that at a total length larger than 80 nm, the scattering of low-wavelength or high-frequency phonons at interfaces occurs. By contrast, at shorter lengths, the wave interference causes a reduction in the amount of thermal conductivity. According to the performed theoretical studies, it can be understood that the minimum infinite thermal conductivity of superlattice nanoribbons is controlled by the superlattice period and the constituent elements of superlattice structures.Several theoretical research works have been performed to unravel the thermal transport in the superlattice nanostructures. For example, the dependency of thermal conductivity of graphene-hBN superlattice nanoribbons on the superlattice period as well as the total length, based on MD simulation, were examinedIn the present work, we evaluate the thermal properties of a series of lateral graphene/graphane superlattice nanoribbons using MD simulations. The study was conducted with various total lengths and superlattice periods, at a mean reference temperature of 300 K and a temperature difference (ΔT) of 40 K between the two sides. Firstly, we verified the effect of the graphene/graphane sample's total length on the thermal conductivity of the superlattice nanoribbon with different superlattice periods (38) were used to investigate the thermal properties of graphene/graphane superlattice nanoribbons having various superlattice periods  with the use of the Nosé-Hoover thermostat and barostatNext, the system was equilibrated via the application of an N.V.T. ensemble  for 1 ns at 300 K.T is the mean reference temperature. Finally, the following expression is used for heat flux (41:2), t is the simulation time, and Finally, the thermal properties of superlattice nanoribbons are computed using the following setup: nanoribbon models are divided into 30 slabs along the X-direction. Atoms present in the left and right edges of superlattice nanoribbon are fixed. The NVE ensemble is applied to the cold and hot boundary slabs region. By applying the N.V.T. ensemble (Nose–Hoover thermostat method), the temperature at the hot and cold slabs is defined as To correctly estimate the considered superlattice's thermal properties, several MD stages must be performed. The employed procedure within our numerical simulations comprises the following steps:After reaching steady-state conditions, the thermal conductivity for a nanoribbon of length L is calculated from Fourier law as follows:g) of 7.1 K. A similar behavior along the interface of a graphene-boron nitride heterostructure for the steady-state temperature is reported by Li et al.42.The superlattice hybrid nanoribbons inevitably contain the interfaces at which the two alternating media meet each other. The presence of these interfaces and their total number affect heat transfer in these nanoribbons. Firstly, we investigate the effect of an individual interface on heat transfer. Figure 43.Figure 44:−11 m2 K W−1.The interfacial thermal resistance at the grain boundary  for two groups of atoms at the left and right sides of the graphene/graphane interface; see Fig. As can be observed, there is a substantial mismatch between the left and right spectrum. This asymmetry of phonon spectra unravels the interfacial thermal resistance and the asymmetrical phonon scattering at the graphene/graphene interface.In this study, MD simulations were applied to study the effect of the total length on the thermal conductivity of graphene/graphane superlattice nanoribbons. At the same time, various superlattice periods (−1 k−1 is observed at In terms of the sample's total length effect, it was observed that an increase in total length leads to elevated thermal conductivity values. However, thermal conductivity at considerable lengths becomes independent from the length and reaches a plateau value. Moreover, two regions of heat transport were identified based on the sample's total length. The turning point between these two regions occurs at the neighborhood of 120 nm, which corresponds to the ballistic-diffusive transition regime. With regards to superlattice period variation, a minimum thermal conductivity of 144 W m


Naloxone is a medication that can quickly reverse an opioid overdose. Syringe service programs (SSPs) are community-based prevention programs that provide a range of evidence-based interventions in the USA, including naloxone distribution. Attributes of SSPs make them ideal settings for naloxone distribution—they have staff and delivery models that are designed to reach people who use drugs where they are. We assessed which outer and inner setting factors of SSPs were associated with naloxone distribution in the USA.n = 263 or 77% of SSPs). We utilized negative binomial regression to assess which factors were associated with the number of naloxone doses distributed and people receiving naloxone.We surveyed SSPs in the USA known to the North American Syringe Exchange Network in 2019. Using the exploration, preparation, implementation and maintenance framework, we assessed inner and outer contextual factors associated with naloxone distribution among SSPs (p < 0.001) and 110% (p = 0.022) higher rate of people receiving naloxone  in the past 12 months. The legal status of SSPs and the level of need was not significantly associated with naloxone distribution. Regarding inner setting, SSPs with proactive refill systems , greater number of distribution days  and older programs  were associated with higher levels of naloxone distribution. Also, SSPs with proactive refill systems ; greater number of distribution days  and older programs  were associated with a higher number of people receiving naloxone.SSPs reported distributing 710,232 naloxone doses to 230,506 people in the prior year. Regarding outer setting, SSPs located in areas with high levels of community support had a higher level of naloxone distribution : 2.09–4.51; We identified outer and inner setting factors of SSPs that were associated with greater naloxone distribution. It is critical to ensure SSPs are adequately resourced to build community support for services and develop service delivery models that maximize naloxone distribution to address the nation’s opioid overdose crisis. Opioid-involved overdose mortality has increased more than six-fold since 1999 with particularly steep increases in recent years . BetweenOpioid-related overdose deaths can be prevented with naloxone—a medication that can quickly reverse the respiratory depression that can be induced by opioids during an overdose . Since tBetween 1996 and 2014, a national study found that over 80% of laypeople who participated in community-based naloxone distribution were PWUD and over 80% of reported overdose reversals were performed by PWUD . A recenSyringe service programs (SSPs) are community-based prevention programs that provide access to sterile syringes and injection equipment . In the While many SSPs pioneered naloxone distribution in the USA, naloxone distribution has not reached sufficient levels of saturation at the community level , 16, 17.The exploration, preparation, implementation and sustainment (EPIS) framework highlights key phases of the implementation process and identifies factors across and within the outer and inner context that can influence the implementation process of evidence-based interventions –24. The Operating since 1989, the North American Syringe Exchange Network (NASEN) has served as a clearinghouse for syringes, supplies, technical assistance and other materials for SSPs throughout the USA. Over the past 30 years, NASEN has served as one of the premier networks and resource centers throughout the USA for SSPs to share knowledge, lessons learned and promotion of best practices. NASEN has one of the most comprehensive datasets of SSPs operational in the USA with (1) the Directory database—SSPs that would like to be listed on their public facing directory; and (2) the Buyers Club database—SSPs that purchase supplies through NASEN at discounted pricing.Our study population included syringe service programs operating throughout the USA in 2019. SSPs were defined as community-based prevention programs that provide access to drug use supplies, including sterile syringes and injection equipment. Though not a requirement, SSPs often provide a range of other services to improve the health of people who use drugs, including overdose education and naloxone distribution; drug testing; vaccinations; testing and linkage to medical care; and other social support services. All SSPs operating within the USA, Territories and Tribal Nations were eligible to participate in our survey. We asked the executive director, or their designee, of the organization to participate in the study by providing information about their SSP.We conducted an online, cross-sectional survey of SSPs throughout the USA from February to June of 2019. The overall goal of the survey was to characterize the outer and inner context of SSPs and their relationship with naloxone distribution. The North American Syringe Exchange Network (NASEN) maintains a database and online directory of SSPs throughout the country which contains contact information and basic organizational characteristics. NASEN contacted all SSPs from their database with an initial email to request their participation in an online survey. Two follow-up emails were sent 2 and 4 weeks after the initial email to encourage participation. For organizations that did not respond to email requests, additional follow-up was conducted with phone calls and one-on-one emails to ensure they had received the communication and could troubleshoot any barriers to their participation. For participating SSPs, the first page of the online survey summarized our study procedures; noted that participation was voluntary; described our provisions to maintain privacy; detailed our reimbursement for their participation; and asked for their consent to participate before they could move forward to provide responses to survey questions. In addition, we used data from the National Center for Health Statistics with regard to the population size and number of opioid overdose deaths in 2017.Our study procedures were reviewed by internal review board within the Office of Research Protection at RTI International’s internal review board and was determined to be exempt under 45 CFR 46.104 (d)(2)(ii). Yet, the study did include a consent process which disclosed the activities of the research, the procedures to be performed, that participation was voluntary, the investigator name and contact information and the provisions to maintain the privacy of subjects. Because the study was determined to be exempt, the consent process did not require a written or electronic signature or a waiver of written documentation of consent. However, participants had to indicate their consent to participate by selecting a button on the webpage before advancing into the online survey.Measures that were included in this study were informed by the exploration, preparation, implementation and maintenance (EPIS) framework . The EPIOur exposure variables included measures from the outer and inner context of SSPs. With regard to the outer context, we asked each SSP about the legal status of their organization—whether they operated in a sanctioned or unsanctioned environment; and how they characterized levels of community support for their program on a scale of 0–100. Community support was dichotomized at the median of the distribution—a local community support rating of 80 out of 100—to allow for enough data to make a comparison and provide a meaningful cut-off point. To estimate the level of need in the surrounding community, we used the opioid overdose mortality rate in the counties where the SSP operated. Collected from the National Center for Health Statistics , these dWith regard to the inner context, we asked each SSP how sustained their OEND implementations were by asking about number of years since they implemented OEND; whether the SSP regularly asked participants if they needed a naloxone refill (proactive) compared to waiting for participants to ask them for a refill (passive); and the number of days of service the SSP provides OEND to their participants in a typical 28-day (4 week) period.p values. We also carried out a sensitivity analysis; first, we log transformed our outcome variables to approximate a normal distribution; then, we utilized linear regression with robust variances to build similar models. All analyses were conducted using Stata (v16.1).We used negative binomial regression models to estimate the association between inner and outer contextual factors on the number of people receiving naloxone and the number of naloxone doses, separately. Models accounted for population size in the county or counties where the SSP operated. We modeled outer and inner contextual factors separately. For each set of analyses, we built full, adjusted models to generate adjusted incident rate ratios (aIRR), 95% confidence intervals (CI) and associated A total of 263 SSPs responded to our survey, representing 77% of the 342 known SSPs operating in 2019. Among the responding SSPs, 247 (94%) indicated that they distributed naloxone, and 237 (96%) of those distributing naloxone provided information with regard to the number of naloxone doses distributed and the number of people receiving naloxone in the prior year. On a scale of 1–100 (with 100 representing high support), the reported median level of community support was 80; the median opioid overdose mortality rate in the surrounding community was 14 per 100,000 people; and most SSPs provided OEND services at least 15 of the last 28 days. In total, SSPs reported distributing 710,232 naloxone doses to 230,506 people in the prior year; per SSP, median annual number of naloxone doses distributed was 880 and median annual number of people receiving naloxone was 350 (Table p < 0.001) higher rate of naloxone distribution and 110% (p = 0.022) higher rate of people receiving naloxone  higher rate of naloxone distribution and 110% (p = 0.022) higher rate of people receiving naloxone. SSPs with a greater number of naloxone distribution days had a 207% (p < 0.001) higher rate of naloxone distribution and 110% (p = 0.022) higher rate of people receiving naloxone. SSPs that had been implementing OEND for a longer period of time had a 207% (p < 0.001) higher rate of naloxone distribution and 110% (p = 0.022) higher rate of people receiving naloxone (Table Regarding the inner context, SSPs with proactive refill systems had a 207% (ne Table . FindingThis study fills an important gap in the scientific literature with regard to understanding relationships of outer and inner contextual factors of SSPs with naloxone distribution. Our most striking finding was that opioid overdose mortality rates in the prior year were not a significant predictor of naloxone distribution. This is particularly concerning as the opioid overdose mortality rates from the previous year are perhaps our best available measure for how much naloxone distribution is needed in a given area. Yet, these findings are consistent with prior research showing that the number of PWID with HIV was not predictive of SSP services , 29. IdeWith respect to outer context, our results showed that SSPs with higher levels of community support in surrounding areas reported higher levels of naloxone distribution. Our findings mirror qualitative findings that demonstrate the pivotal role that surrounding communities can play either through support or resistance . Taken tOur results also showed that unsanctioned SSPs had similar levels of naloxone distribution compared to those operating in sanctioned environments. Prior research has shown that policy/legal environments are important predictors of whether community-based naloxone programs or buprenorphine treatment within SSPs are implemented , 34. TheSupporting programs to increase their days of service and supporting them into sustainment is important for building larger-scale naloxone distribution. While our study did not collect information about funding levels, recent studies have shown that the median annual budget of SSPs was $85,000 in 2020 , 35. ThiOur finding that proactive refill systems were associated with higher levels of naloxone distribution highlight an important, modifiable organizational characteristic. A recent initiative highlighted that strengthening refill systems can be a critical component to improving the number of doses and people with naloxone in the community . SSPs haFor this study, the EPIS framework was an important tool to strengthen our understanding of organizations’ outer and inner contextual factors and their relation to the scale of naloxone distribution . A recenIt is important to consider potential methodological limitations with our study. We are unable to generalize to all SSPs in the USA as we do not know the full universe of SSPs. Since 1992, NASEN has served as the primary resource for SSPs in the country. NASEN operates a buyers club for SSPs to acquire supplies for harm reduction services, offers free supplies to new and resource-constrained SSPs, and maintains a database of all known SSPs in the USA. We utilized this information to get in touch with SSPs for our survey, but it remains possible that other SSPs are unknown to NASEN. Since 23% of SSPs did not complete the survey, there is also the possibility of response bias. It is particularly plausible that underfunded or smaller SSPs had a lower response rate . In addiThis study identified outer and inner setting factors were associated with greater naloxone distribution from syringe service programs. With rising opioid overdose mortality rates for over 2 decades, the need to address these factors is of paramount importance, and past due. Ensuring that SSPs operate within supportive legal and community environments; receive sufficient and sustained financial support and build strong delivery systems is vital to maximize naloxone distribution in such a way that effectively addresses the nation’s opioid overdose crisis.


It is proposed that nanosized graphene aggregation could facilitate coherent neutron scattering under particle size conditions similar to nanodiamonds to enhance neutron intensity below cold neutrons. Using the RIKEN accelerator-driven compact neutron source and iMATERIA at J-PARC, we performed neutron measurement experiments, total neutron cross-section and small-angle neutron scattering on nanosized graphene aggregation. For the first time, the measured data revealed that nanosized graphene aggregation increased the total neutron cross-sections and small-angle scattering in the cold neutron energy region. This is most likely due to coherent scattering, resulting in higher neutron intensities, similar to nanodiamonds. Slow neutrons, such as cold neutrons, are excellent for observing light elements such as hydrogen. They are also important nondestructive probes not only for basic physics but also for the structural genomics advancements in the life sciences and the battery technology advancements needed for the transition to a hydrogen society. Neutron-based science, also known as high-intensity-dependent science, demands that we increase the source’s intensity as high as possible. Cold neutrons are generally generated by cooling neutrons produced by nuclear reactions, spallation reactions based on accelerators, and fission reactions in nuclear reactors to a thermal equilibrium state with a hydrogenous material such as solid methane.2) [However, it is not easy to obtain slow neutrons below thermal equilibrium using this method, such as ultra-cold neutrons or very-cold neutrons. A new unique method focusing on nanosized particle aggregation has been proposed to increase neutron intensity in that energy region ,2,3,4,5.2)  have recWe focused on another carbon structure, graphene, to find a solution to this problem. Graphene ,13,14 isConversely, in its development progress, large amounts of nanosized end materials are also being produced. We hypothesized that nanosized graphene could aid coherent neutron scattering under particle size conditions similar to nanodiamonds. Moreover, it might be possible to use it in high neutron radiation conditions due to graphene’s strong sp2 bonds. Using the RIKEN accelerator-driven compact neutron source (RANS)  and iMATIt is also difficult to determine how much useful information can be obtained from a 700 W RANS small power source, such as total cross-section and small-angle scattering measurements, when the source intensity is overwhelmingly disadvantageous compared to MW class high-power sources like those at J-PARC . As a reIn this paper, we report the potential of nanosized graphene as a reflector material below cold neutrons, together with experimental results.A combination of phenolic formaldehyde resin carbonization and the hot isostatic pressing (HIP) process produces a distinctive graphene called a “graphene flower” with a “sunflower” shape ,23. NoteFor this study, two graphene sample types, namely, graphene 1 and 2, were prepared under different HIP conditions. First, a phenol formaldehyde resin, BellPearl S870, manufactured by Air Water Co., Ltd., with an average particle size of 15 μm, was prebaked in a constant flow of nitrogen gas at a maximum temperature of 650 °C. The heat-treated powder was placed in a graphite crucible and subjected to HIP in argon gas at a maximum ultimate pressure of 70 MPa. Graphene 1 was heated to a maximum temperature of 1390 °C at a rate of 900 °C/h, held there for 1 h, and then cooled. Graphene 2 was heated to a maximum temperature of 1300 °C at a rate of 200 °C/h and then cooled immediately without any retention time.As shown in 4C) slits in the locations shown in The experiment was carried out using the RANS neutron source, which was generated by the p-Be reaction of a 7 MeV proton bombardment, as shown in Neutron transmission was measured using the two-dimensional neutron detector with Gas Electron Multiplier, called an n-GEM  detectorAlthough this is only a relative comparison due to the different moderator and detector positions, the mesitylene moderator’s cold neutron component, forming a peak at 3–5 meV, was confirmed.SANS experiments were performed at the J-PARC iMATERIA ,18. MeasCompared to graphite, nanodiamond had several hundred higher total neutron cross-sections below the meV region. Graphene 1 was approximately one-third of the total neutron cross-section data of nanodiamonds. Conversely, it was several tens of times higher than graphite. The findings indicate that nanosized graphene, which is still in the early development stages, has the potential to rival or even surpass nanodiamonds with further fabrication process optimization, including nanosized graphene aggregation.−1 for SANS obtained for graphenes 1 and 2, originating from interlayer interference. The SANS profiles obtained for graphenes 1 and 2 showed broad shoulders at q = 0.15 Å−1, attributed to a graphite primary particle sheet [−4, indicating a smooth surface. Compared to nanodiamonds, and graphenes 1 and 2, the SANS obtained for graphite showed a strong upturn obeying q−4 toward lower q = 0.01 Å−1, indicating a larger particle sheet size. It should be denoted that for graphenes 1 and 2, we could not recognize the fringes or oscillations in the q-behavior. This is due to the large inhomogeneity in shape and size of the graphenes.le sheet . The shole sheet , the tai−1, the q-behavior was consistent with that obtained at the iMATERIA. The ib-SAS instrument is still being developed to reduce the noise background.The SANS profile of graphene 1 was also measured using the RANS-developed small-angle scattering instrument (ib-SAS ) see op. Even thQ, representing the scattering cross-section due to small-angle scattering [−1. Q values. In particular, nanodiamond and graphene 1 showed a larger Q. These findings agree with the scattering cross-section values determined by transmission experiments.Invariant attering , was evaWe concentrated on nanosized graphene aggregation to intensify below cold neutrons, which are similar to those of nanodiamonds. Using the RANS, we performed neutron measurement experiments, total neutron cross-section, and SANS on nanosized graphene aggregation. For the first time, the measured data revealed that nanosized graphene aggregation increased total neutron cross-sections and small-angle scattering in the cold neutron energy region, most likely due to coherent scattering, implying the possibility of below cold neutron applications. We will go on to focus on the radiation resistance of graphene, which is expected from the strong sp2 bond.


Scientific Reports 10.1038/s41598-023-28617-w, published online 24 January 2023Correction to: The original version of this Article contained an error in the legend of Figure 1.21 W/cm2 peak intensity, ionizes a 1.5 μm-long (y) and 1.2 μm-thick (x) stack of graphene layers. The interaction results in self-injected electrons being accelerated to ≃ 7 MeV. The image is at scale, with a 150 nm bar drawn, and for better visibility, only 15 out of 60 graphene layers are shown. The simulated normalized transverse electric field (Ex) is shown as a surface colour plot for the same laser pulse before entering the target (left) and after leaving the target (right). This work contains 2D PIC simulations carried out in the yx-plane indicated in the image.”“Overview of the catapult electron acceleration scheme in graphene layers. Moving from left to right, as indicated by the blue arrows, a single 3 fs-long laser pulse of 100 nm wavelength and 10now reads:21 W/cm2 peak intensity, ionizes a 1.5 μm-long (y) and 1.2 μm-thick (x) stack of graphene layers. The interaction results in self-injected electrons being accelerated to ≃ 7 MeV. The image is at scale, and for better visibility, only 15 out of 60 graphene layers are shown. The simulated normalized transverse electric field (Ex) is shown as a surface colour plot for the same laser pulse before entering the target (left) and after leaving the target (right). This work contains 2D PIC simulations carried out in the yx-plane indicated in the image.”“Overview of the catapult electron acceleration scheme in graphene layers. Moving from left to right, as indicated by the blue arrows, a single 3 fs-long laser pulse of 100 nm wavelength and 10The original Article has been corrected.


Candida albicans and Streptococcus mutans and the surface roughness of a 3D-printed polymethylmethacrylate dental resin enhanced with graphene. A 3D-printed polymethylmethacrylate dental resin was reinforced with four different concentrations of graphene: 0.01, 0.1, 0.25 and 0.5 wt%. Neat resin was used as a control. The specimens were printed in a liquid crystal display printer. Disc specimens were used in antimicrobial evaluation, and bar-shaped specimens were used to measure surface roughness. The study of antimicrobial activity included the inhibition of the growth of C. albicans and S. mutans and their adhesion to the resin’s surface. Surface roughness increased with the increase in the graphene concentration. The growth inhibition of C. albicans was observed in the different concentrations of graphene after 24 h, with no recovery after 48 h. The specimens doped with graphene were capable of inactivating S. mutans after 48 h. The surface-adhesion studies showed that the density of microbial biofilms decreases in the case of specimens doped with graphene. Graphene, despite increasing the resin’s surface roughness, was effective in inhibiting the growth and the adhesion to the resin’s surface of the main inducers of prosthetic stomatitis.The present study aimed to test, in vitro, the antimicrobial activity against  Up to the middle of the 20th century, the great majority of the elderly was edentulous. However, during the last few decades, due to an improvement in dental care combined with a professional oral care approach that follows the principles of prevention and minimally invasive dentistry, a reduction in the prevalence of edentulism and the incidence of tooth loss has occurred in many industrialized countries . Therefore, despite the improvement in oral health care, the increase in life expectancy leads to prosthetic rehabilitation continuing to be a need with high prevalence. The World Health Organization (WHO) predicts that in 2050, the percentage of the population that is over 60 years of age will be close to 30% in developed countries [Polymethylmethacrylate (PMMA) is still a clinician’s first choice for various prothesis fabrications: denture bases, fabrication of artificial teeth, temporary crowns, obturators for cleft palates and denture relining and repair. A drawback of conventional denture acrylic resins is the high susceptibility to microbial colonization in the oral environment due to some properties such as porosity, surface charge, amount of surface free radicals, hydrophobicity and surface roughness ,4. So, uSome studies ,8 have dThe adhesion of microorganisms to hard dental surfaces is followed by the formation of plaque . SurfaceDenture stomatitis is a very common disease that affects denture users. It has a high prevalence and may affect, according to some studies, two-thirds of removable denture wearers ,12,13. ACandida albicans is an opportunist fungus found in human mucosa, but, under local or systemic conditions, it might invade tissues and become harmful. It is responsible for denture stomatitis in 50% to 90% of infections, but other species are also present, such as C. tropicallis or C. glabrata [glabrata ,17. Candida spp. but of multispecies biofilms that may include Streptococcus mutans and Staphylococcus aureus. S. mutans is often found in the surface of acrylic resins and when incubated together with C. albicans, can compete for biding sites, but it also may favor yeast adhesion [Studies have shown that denture stomatitis is not only a result of adhesion .The increasing use of antifungal drugs, such as nystatin, fluconazole, itraconazole or chlorhexidine, in the treatment of denture stomatitis has shown some serious side effects and, additionally, has led to the development of resistant fungal and bacterial strains ,20. 2) and silver nanoparticles, when mixed with polymers. Nanoparticles have a higher level of fungicidal effects than conventional antifungal medicines as they have better penetration in host cells and tissues, even in small concentrations [To overcome these problems, recent research has been carried out by incorporating nano additives, in the form of nanospheres, nanosheets, nanofibers or nanotubes, to the denture base resin. Recent studies have proven antifungal efficacy of various nano materials, such as zirconium, magnesium oxide (MgO), zinc oxide (ZnO), titanium oxide  and graphene oxide reduced (rGO), is very promising. There is evidence that graphene has the ability to reduce the viability of microorganisms in the oral cavity. The mechanism of antimicrobial action occurs by damage in the cell wall followed by cell lysis . CAD/CAM technology is an increasingly evident reality in Dentistry. 3D printing, or additive manufacturing, is a technology that allows a quick planning and printing of 3D objects, so it is expected to be of great value in the prosthetic rehabilitation clinical practice. This manufacturing method involves adding layers of resin material incrementally, with light activation, to build the prothesis in the process. The new 3D printing technology leads to the development of new biomaterials such as PMMA photocurable resins whose properties need to be improved for biomedical applications ,27. A waCandida albicans and Streptococcus mutans. The null hypothesis stated that there was no significant difference in the surface roughness and antimicrobial effect of PMMA with and without graphene incorporation.The aim of this study was to evaluate the effect of graphene nanoplatelets added to a 3D-printed PMMA resin on surface roughness and antimicrobial activity against Graphene nanoplatelets (GNP)  with 8–30 layers, a thickness of 3–10 nm and layers’ lateral dimensions of 0.5–2 µm were added to Dental Sans resin , which is the commercial name for a photosensitive liquid mixture of methacrylate oligomers and monomers. The graphene concentrations were: 0.01 wt%, 0.1 wt%, 0.25 wt% and 0.5 wt%. The resultant mixture was homogenously mixed to ensure the full dispersion of the nanomaterial, with a magnetic stirrer for 45 min at a setting of 1500 rpm. The viscosity of the resins, reinforced with the different graphene concentrations, was tested, and this property did not suffer significant changes with the presence of graphene, remaining at the value of 1.2 Pa.s. To characterize the antimicrobial activity of the graphene–PMMA resin, a total of 70 cylinder-shaped specimens  were used and divided into 5 groups: control group (Gc) and 4 experimental groups according to the different concentrations of graphene . A total of 25 bar-shaped specimens (50 × 10 × 4 mm) were used to measure surface roughness, 5 for each group.A CAD software Solidworks  was used to virtually design the specimens according to the above-mentioned dimensions . The CADThe specimens were 3D-printed using a liquid crystal display (LCD) technology , which is equipped with a 405 nm light-emitting diode (LED) as the light source to cure the liquid polymer resin layer by layer. The fabrication process is illustrated in 3, and the viscosity was 1.2 Pa.s. The addition of graphene did not appreciably change these properties of the polymer.The printing direction was 45 degrees. The layer resolution was 0.05 mm for all samples, and the exposure time was 1.9 s. The density of the printed material was 1.20 g/cmAfter printing, all the specimens were cleaned using a 90% isopropyl alcohol bath in a postprocessing machine  for 5 min to remove the excess liquid resin according to the manufacturer’s indications. Then the specimens were placed in an oven at 80 °C for 30 min  for post-print baking. Postpolymerization was carried out in a UV light curing unit for 10 min . The support structures were removed after post curing using low-speed rotary instruments (5000 rpm) .The specimens were then finished, polished and measured by using a digital caliper with a 0.01 mm resolution , ensuring that the specimens conformed to the dimensional tolerances of the ISO 20795-1. After measurements, all specimens were disinfected by using 70% ethanol for 5 min and were stored in 37 °C water for 24 h according to ISO 20795-1.Raman spectroscopy was carried out to identify the presence of GNP in the polymer resin at different concentrations. Measurements were carried out on an ALPHA300 R Confocal Raman Microscope  using 532 nm laser light for excitation at room temperature . The laser beam was focused on the sample by a 100× lens. Single acquisitions  were performed using the 600 g/mm grating. The surface roughness profile for each specimen was measured using a contact profilometer . The surface of the specimen was covered by a 5 μm ray diamond tip with constant load following a rectilinear measurement length of 4.8 mm for 10 s. Three measurements were performed for each specimen. The average roughness (Ra) was analyzed. Ra (µm) is the arithmetic mean value of all heights  in the given roughness profile.Candida albicans ATCC 11225, stored at −80 °C in Sabouraud Dextrose broth  with 20% glycerol, and Streptococcus mutans DSM 20523, stored at the same conditions in Trypticase Yeast Extract broth , were used.In this study, S. mutans was subcultured in Trypticase Yeast Extract agar , and, prior to the experiments, one isolated colony was inoculated in 10 mL of Yeast Extract broth  and grown anaerobically at 37 °C overnight. An aliquot of each culture (300 μL) was transferred into a new fresh liquid medium and grown under the same growth conditions until stationary growth phase was achieved.The fungus was subcultured in Sabouraud Dextrose agar , and, before each assay, one isolated colony was inoculated in SDB and grown aerobically at 37 °C overnight at 170 rpm. 8 colony-forming unit per milliliter (CFU/mL) (corresponding to 0.5 Macfarland). The diluted microbial suspensions were equally distributed in sterilized microtubes (1 mL per tube) containing each graphene–PMMA resin specimen. A microbial control without any disk (Ct), to evaluate the growth profile of each microorganism over time, and a resin control (Gc) (nondoped with graphene), to evaluate the antimicrobial effect of PMMA resin, were also included. The suspensions were incubated at 37 °C under a stirring rate of 120 rpm. To evaluate the growth inhibition effect of the specimens, the CFU/mL was determined. Thus, aliquots of samples and controls were collected at times 0 h, 24 h and 48 h of incubation; serially diluted in Phosphate-Buffered Saline ; and pour plated in triplicate in SDA for C. albicans and ISP agar for S. mutans. Prior to serial dilutions after the incubation period, all microtubes were sonicated for 1 min. The Petri plates were incubated at 37 °C for 24 h (plates containing S. mutans were incubated at the same temperature in anaerobic chambers), and the viable microbial density was expressed as Log CFU/mL. Three independent experiments, with two replicates (n = 2) for each condition, were performed for each assay. The average of the results was calculated.Previous to each assay, the graphene–PMMA resin specimens were submitted to UV light for 30 min to avoid contamination. For the bioassay purposes, each microorganism culture, grown overnight and in stationary phase, was diluted tenfold in culture medium to a final concentration of ~10C. albicans and S. mutans that were subcultured according to the previous conditions were equally distributed in a 96-well microplate (150 μL per well) containing each sterilized graphene–PMMA resin specimen. A resin control (Gc) (nondoped with graphene) was included. The suspensions were incubated at 37 °C under a stirring rate of 120 rpm for 48 h. The samples were fixed in 2.5% glutaraldehyde in PBS for 40 min at RT. After three washes in PBS, the samples were dehydrated through a graded ethanol  series  [n = 3).The microbial cultures of in each) ,30. The p value < 0.05 was indicative of statistical significance.Statistical significance of surface roughness was determined by Kruskal Wallis analysis of variance with a post hoc test (Bonferroni) using Statistical Package for the Social Sciences . A p < 0.0001 was considered statistically significant.The statistical analysis of the antimicrobial activity was performed using GraphPad Prism 9.0.0 . The results were presented as mean of at least 3 independent assays with 2 replicates per condition and the respective mean ± standard deviation. The significance of microbial concentration between graphene–PMMA resin specimens, and along the experiments, was assessed by two-way ANOVA analysis of variance. Tukey’s multiple comparison test was used for a pairwise comparison of the means. The significance of differences was evaluated by comparing the results obtained in the test samples with results obtained for the control (Ct) at different times. A value of −1, and 2D (~2690 cm−1), a second overtone of a different over plane vibration, D (~1350 cm−1).The Raman spectroscopy results showed that the graphene platelets consist mainly of few-layer graphene and that photo-polymerized resin is largely PMMA. Raman spectroscopy was performed to identify and evaluate the quality and structural properties of the graphene according to the intensity, frequency and linewidth of its G, D and 2D Raman-modes. The Raman spectra of graphene are characterized by two main peaks: the primary in-plane vibrational mode, G  and 0.5 wt% (p = 0.005) in comparison with neat resin. In the lower concentration (0.01 wt%), the resin’s surface roughness was approximately the same as neat resin.Surface roughness was evaluated , and it C. albicans and the Gram-positive bacteria S. mutans. The different graphene–PMMA specimens, corresponding to different percentages of graphene, were incubated with each microorganism, and the growth inhibition effect was evaluated after 24 and 48 h of exposure. The results are presented in C. albicans and (b) for S. mutans.The antimicrobial activity of each graphene–PMMA specimen was evaluated against two common microorganisms present in oral biofilms: the yeast C. albicans (p = 0.7213). However, the growth inhibition of C. albicans was observed when this fungus was incubated with the specimens doped with different percentages of graphene for 24 h , with no significant recovery after 48 h of incubation. It was also observed that there were no significant differences amongst the graphene–PMMA doped with different percentages of graphene. These results made it clear that not only does graphene exert the growth inhibition of C. albicans, but also that the antimicrobial activity effect of the graphene–PMMA does not depend on the graphene amount. The results showed that when albicans a was incS. mutans, the antimicrobial effect of the graphene–PMMA specimens had promoted a different profile (p > 0.9999). In the case of specimens doped with 0.01, 0.1 and 0.25 wt% graphene, this effect was not observed in the first 24 h; however, after 48 h of incubation, the bacterial survival rate decreased in the c.a. of 1.5 log CFU/mL when compared to the initial time. In what concerns the specimens containing 0.5 wt% of graphene, the growth inhibition of S. mutans was observed after 24 h of incubation , and after 48 h, the bacterial inactivation of 1.6 log CFU/mL was achieved . These results suggest that PMMA specimens doped with graphene are capable of inducing the inactivation of S. mutans after 48 h of exposure. Regarding  profile b. As in C. albicans and the Gram-positive bacteria S. mutans biofilms on different graphene–PMMA specimens with different percentages of graphene was studied by SEM. Thus, each microorganism was incubated in the presence of each specimen for 48 h. This incubation time was chosen according to the antimicrobial activity studies. C. albicans and S. mutans biofilms, respectively.The surface adhesion of the yeast C. albicans and S. mutans was more pronounced in the graphene-doped-PMMA resins  was compared with the graphene–PMMA resin disks. These results are in accordance with the ones achieved in the microbial growth inhibition studies where the inhibition growth of A resins . PMMA is a dental material widely used and is still a clinician’s first choice for several prothesis fabrications. Despite its biocompatibility, reliability, ease handling and low toxicity, this polymer has poor antimicrobial activity which contributes to several oral infections ,32. To oC. albicans after incubation for 48 h. Moreover, the graphene–PMMA specimen at a higher concentration (0.5 wt%) had not only inhibited the S. mutans growth after 24 h of incubation, but also had inactivated this bacterium after 48 h, showing its bactericide properties. The same bactericide effect was afforded for all the remaining graphene concentrations after 48 h of incubation. The surface adherence studies were in line with these results once the density of microbial biofilms on the graphene–PMMA specimen decreased with the increase in the graphene concentration in the graphene–PMMA resin disks. Herein, 3D-printed PMMA dental resins, reinforced with different concentrations of graphene , were prepared and characterized, and their antimicrobial activity against two common microorganisms present in oral biofilms and their microbial adhesion properties were studied. We had demonstrated that the surface roughness of the graphene–PMMA specimens increased with the increase in graphene concentration. However, despite the increase in surface roughness, graphene proved to have an antimicrobial activity. It was clear that PMMA doped with different concentrations of graphene inhibited the growth of C. albicans after incorporating nGO than those exhibited by pure PMMA via increased hydrophilicity, which envisage the potential usefulness of graphene as a promising antimicrobial dental material for dentures, orthodontic devices and provisional restorative materials. Although the approach for PMMA resins doped with graphene preparation is not the same, some studies regarding their antimicrobial activity and/or the adhesion properties of PMMA–graphene hybrids were reported. Lee et al.  reportedC. albicans and for the inactivation of S. mutans after 24 and 48 h of incubation, respectively, there are no significant differences in the graphene concentration of the PMMA–graphene specimen, with a necessary minimum concentration of 0.01 wt% of graphene to exert those effects. In 2019, Said et al. [S. mutans and the thermal expansion of commercially available PMMA incorporated with nano graphene oxide in different concentrations . In what concerns the antimicrobial effect of these hybrids, 0.5 wt% of graphene oxide was the minimal inhibitory concentration achieved for inhibiting the growth of S. mutans. In a similar work, the antimicrobial properties against E. coli and P. aeruginosa of PMMA fibers containing 2, 4 and 8 wt% of graphene nanoplatelets were studied. [C. albicans of acrylic-based tissue conditioner doped with 1, 3 and 5 wt% of nano graphene oxide, the growth inhibition of C. albicans was observed only for the highest concentration of graphene oxide [Our results suggested that for the inhibition of d et al.  reportedstudied.  The resune oxide . The facne oxide ,44. It iC. albicans and S. mutans. The results seem to demonstrate an increase in surface roughness of PMMA–graphene disks with the increase in graphene concentration. Additionally, antimicrobial activity against C. albicans and S. mutans was achieved for the specimens doped with graphene. Surface adhesion studies corroborate that these materials have anti-adhesive effects against the two main microorganisms responsible for prosthetic stomatitis. These findings open the perspective of the use of 3D-printed PMMA–graphene materials in medical and dental applications, namely in denture prosthetics, but future investigations are needed to understand the mechanism of action of PMMA-graphene disks.This study reported the effect of GNP added to a 3D-printed PMMA resin on surface roughness and the antimicrobial activity against


At negative voltages, the GPETNC serves as a mono/divalent ion selective device by impeding most divalent cations to transport through; at positive voltages, it mimics a biological K+ nanochannel, which conducts K+ much more rapidly than the other ions with K+/ions selectivity up to about 4.6. Besides, the GPETNC also exhibits the promise as a cation-responsive nanofluidic diode with the ability to rectify ion currents. Theoretical calculations indicate that the voltage-dependent ion enrichment/depletion inside the GPETNC affects the effective surface charge density of the utilized graphene subnanopores and thus leads to the electrically controllable ion sieving. This work provides ways to develop heterogeneous nanochannels with tunable ion selectivity toward broad applications.Ion-selective nanoporous two-dimensional (2D) materials have shown extraordinary potential in energy conversion, ion separation, and nanofluidic devices; however, different applications require diverse nanochannel devices with different ion selectivity, which is limited by sample preparation and experimental techniques. Herein, we develop a heterogeneous graphene-based polyethylene terephthalate nanochannel (GPETNC) with controllable ion sieving to overcome those difficulties. Simply by adjusting the applied voltage, ion selectivity among K Nanoporous 2D materials have shown promising potential for ion sieving applications due to their physical and chemical properties. Here authors develop a heterogeneous graphene-based polyethylene terephthalate nanochannel with ion sieving ability that is controlled by adjusting the applied voltage. The boom of 2D materials such as graphene and molybdenum disulfide (MoS2) makes nanoporous atomically thin membranes (NATMs) highly promising as next-generation ion-selective membranes, owing to their ultra-thin material thickness, high mechanical strength, and great chemical robustness8. These nanochannel devices are supposed to have different ion selectivity in different situations. In energy harvesting scenario, it is crucial for ion-exchange membranes with high cation/anion selectivity to achieve the high conversion efficiency and output power density10. Rapid and selective conduction of single ions  is concerned in a biomimetic ion transport channel12. The industry of lithium-ion batteries needs efficient extraction of Li+ from brine or seawater with the exclusion of other ions14. Under each circumstance, a corresponding device with specific ion selective nanochannels is required to be fabricated, which could be time- and resource-consuming and limited by experimental techniques.Ion selective artificial nanochannels have recently attracted general interest in water desalination23. For example, Abraham et al.15 prepared graphene oxide (GO) membranes with different interlayer spacing by adjusting the relative humidities of the sealed storage containers, and those GO sheets exhibited different relative cation permeation rates. Using a time-dependent nitrogen-doping technique, Song et al.16 created several nitrogen-doped nanoporous graphene with different ion sieving abilities. Zhang et al.17 reported that the GO membranes with controllable surface charges introduced by polyelectrolyte modification exhibited various ion selectivity and charge-guided ion transport. Also, Sun et al.18 fabricated hybrid GO-based membranes with different surface charges to achieve different charge-sensitive sieving of cations. However, in those works, a single functional membrane was actually fabricated in each case, and its ability to sieve ions was still untunable, which is the same for NATMs with diverse structures  created using different methods21. A nanochannel device with pH- or electrical-tunable ion selectivity could be promising29. Some nanoporous membranes had pH-dependent ion selectivity possibly due to pH-sensitive electric charges23, but their performances might degrade in the case of given solutions with fixed pH value and they could not fast tune the ion selectivity for some industry demands. Although controllable water permeation and ion flow through nanochannels could be realized by electrically gating29, experimental research on ion selectivity is rare and the mechanism remains elusive .NATMs with tunable ion selectivity are desirable to overcome those difficulties and have gained widespread attention in the last few years31. In a rectifying nanochannel, such as a conically shaped polyethylene terephthalate nanochannel (PETNC), which is promising as a biomimetic nanochannel due to its flexibility and ease of chemical modification, the cation/anion selectivity can be achieved34, but it is difficult for a conical PETNC with entrance diameters larger than several nanometers to sieve metal ions37, because such sizes are much larger than the ion hydrated diameters (a few angstroms38) in electrolyte solutions and therefore the factors leading to ion separation 3 hardly work. Nevertheless, the voltage-dependent ion transport in a rectifying nanochannel suggests that a nanochannel device with heterogeneous structure consisting of the pristine 2D nanopores with unchangeable ion selectivity along with the rectifying nanochannel might achieve voltage-tunable ion sieving. Such studies are few because NATMs are usually supported by non-selective and non-rectifying nanoporous materials such as SiNx and anodized aluminum with μm-sized cylindrical nanochannels, which are merely supports with negligible influence on the ion sieving8.Ion flow can also be tuned by applied voltage in a nanochannel with ion rectification, which is the directional transport of the ions through the channel at different voltages+, Na+, Li+, Ca2+, and Mg2+, which is capable of quickly meeting different demands for complex and variable scenarios in practice. The GPETNC is also able to rectify ion currents with opposite rectification for mono- and divalent ions. Finite element calculations indicate that the tunable ion selectivity arises from the different effective surface charges of graphene subnanopores influenced by the voltage-dependent enrichment/depletion of ions inside the GPETNC. The opposite preferential transport directions of the mono- and divalent cations arise from the differences in the diffusion currents, which are generated by the ion concentration gradients. The multifunctional GPETNC exhibits its promise as not only a mono/divalent ion selective device but also a biomimetic ion channel, as well as a cation-sensitive diode, which indicates the great potential of such intriguing heterogeneous nanochannels for diverse applications.Here, we design a heterogeneous GPETNC with graphene subnanopores on the base side of a conical PETNC. By taking the best of both the components, it exhibits excellent voltage-controlled ion selectivity among K8. An asymmetric track-etching technique40 was used to fabricate the conical nanochannel in a 12-μm-thick PET membrane. Briefly, a pristine PET membrane was first irradiated by a single swift heavy ion, which could create a nanometer-sized damage track in the membrane, and then asymmetrically etched with one side of the membrane contacting the etchant and the other contacting the stop medium. The fabricated conical PETNC had the base entrance diameter of ∼840 nm estimated from the etching time and the tip diameter of ∼140 nm obtained by fitting the size of a theoretical model to experimental data  was used to characterize the graphene subnanopores created under the ion irradiation, and the pores had the average diameter of ∼0.5 nm and density of ∼5.47 × 1011 cm−2  curves in a custom-designed system with two cells filled with chloride salt solutions using Ag/AgCl electrodes , which does not explicitly depend on the channel size and has been successfully applied in studying the cation/anion selectivity of graphene nanopores43. The high selectivity ratio confirmed that the transport of Cl− through the GPETNC could be neglected compared to that of cations.To measure the ion selectivity, the GPETNC was characterized by recording the current-voltage , which was the measured ion conductance in 0.1 M cation-chloride solutions  adjusted to consider the differences in electric mobilities and charges of the cations (as shown in “Methods”). The selectivity Si defined using this method is effective to describe the ability of the GPETNC to sieve different ions (a larger Si indicates that cation i can transport more easily) when the ion current caused by Cl− is negligible43. Figure I–V curves of the GPETNC in different chloride salt solutions, which are nonlinear and different from the results of pristine graphene nanopores43. Therefore, the normalized conductance gi was calculated at different voltages . In addition, the ion selectivity was voltage-dependent, especially for monovalent ions (Na+ and Li+), whose selectivity decreased with the increase of applied voltage.The ion selectivity of the GPETNC is presented in Fig. +/ions selectivity of about 4.6 at 0.2 V  could be accomplished by the GPETNC while many devices fail in this. The results indicate that the great promise of the GPETNC as a versatile ion selective nanochannel because its ion selectivity can be quickly adjusted by adjusting the applied voltage, with no need in additional modifications to the sample itself. Furthermore, the GPETNC suggests such a heterogeneous nanochannel, in which, for example, nanoporous 2D materials such as MoS2, hBN, GO, and early transition-metal carbides and carbonitrides (MXenes) combined with the conical PETNC, might also exhibit a facilely tunable ion sieving ability.We further compared the ion selectivity of the GPETNC with that of some NATMs reported before54 or mobility57, which is characterized by the measured ion current, it is difficult to study their performance in mixed solutions, because all ions contribute to the total current and their portions are hard to be distinguished. Taking K+ and Na+ as an example, we fabricated a GPETNC with ion selectivity shown in Supplementary Fig. I–V curve in the mixed solution containing both K+ and Na+ was complicated and indicated the elusive effect of the interaction between K+ and Na+ on the ion transport  ion from a mixture of ions. Therefore, such research is valuable for each emerging ion selective nanochannel. However, for nanochannels with selectivities based on the experiments of ion conductanceI–V curves of the GPETNC in different chloride salt solutions , and its value also reflects the preferential direction of ion transport in rectifying nanochannels well59. Figure +, Na+, and Li+), their rectification ratios were smaller than 1, which indicated that the transport of these ions in the GPETNC had a preferential direction from the tip side to the base side. However, for divalent ions (Ca2+ and Mg2+), the preferential direction of transport reversed as base-to-tip, as the rectification ratios were found to be larger than 1. Although ion rectification phenomenon exists in a pristine conical PETNC in different chloride salt solutions at the same concentration37, the rectification ratios are always smaller than 1 for not only monovalent ions but also divalent ones, which indicates that the preferential direction of all of them keeps from the tip to the base entrance. Besides, the preferential direction does not reverse in electrolyte solutions with different concentrations for a pristine conical PETNC37, and neither did the GPETNC 8, which we speculated to be affected by the voltage-dependent enrichment/depletion of ions inside the GPETNC to some extent.The performance of a nanochannel can be attributed to its structureTNC Fig. . The ionTNC Fig. . The mai68, including not only PETNCs66 but also nanopores in 2D materials68.To test the hypothesis, we calculated the distributions of the ion concentration and electric potential in the GPETNC in chloride salt solutions at different applied voltages by numerically solving the Poisson-Nernst-Planck equations (Eqs. –6) in “ in “6) i66. z = 12 μm), which indicates that a great number of K+ accumulated around there .Our theoretical calculations indicated that indeed there were the voltage-dependent enrichment and depletion of ions inside the GPETNC, and the distribution of the potassium ion concentration ere Fig. b owing tψ around there46. As shown in Fig. ψ far away from the graphene is close to the applied voltage, and it decreases as the location approaches the graphene surface because of the negative charges. For the same electrolyte solution, the effective charge density of graphene surface ψ between the value of ψ far away from the graphene surface and the value on the surface; a larger Δψ indicates a higher 46. Figure ψ in the outer side of the graphene surface barely changed at different voltages, because of the nearly unchanged degree of cation enrichment there . Therefore, the inter-cation selectivity of the GPETNC relied on the applied voltage due to the voltage-dependent ψ of the GPETNC at −1 V was much higher than that at 1 V 3. Such investigations are beyond the scope of this work, and thus future research can, for example, focus on the reasons why the experimentally measured ion selectivity of the GPETNC did not monotonously rely on the voltage, as departed from Δψ in theoretical calculations.The ion selectivity of the GPETNC arose from its graphene subnanopores as discussed before. It has been reported that the value of charge density of graphene nanopores influences the ion selectivityentclass1pt{minimaons Fig. . Hence, entclass1pt{minima+/Cl− selectivity of 46 as demonstrated before, which indicates that the transport of cations mainly contributes to the ion current. Theoretical calculations indicated the existence of the voltage-dependent cation enrichment/depletion inside the GPETNC , suggested by our work, might also have a voltage-tunable ion selectivity even with a wider range and the cation-sensitive ion rectification with a more excellent rectification ratio. The creation of such composite can be facile and simple, because of the mature fabrication techniques of NATMs42 using a graphene CVD system . The Cu foil was successively cleaned via sonication in deionized water, acetone, and isopropyl alcohol (IPA) for 5 min to remove surface contaminants. After dried, the Cu foil was folded as a pocket with the open edges crimped to improve the quality of synthesized graphene in the interior surface of the pocket77. In the graphene growth, the Cu pocket was placed in a tube furnace and heated to 1030 °C for 30 min and maintained the temperature for 5 min in a mixture of 10 sccm H2 and 10 sccm Ar. Then, 10 sccm CH4 was additionally introduced for 20 min under a total pressure of ~0.30 Torr. The furnace was subsequently cooled down to ambient temperature in the mixture of 10 sccm H2 and 10 sccm Ar for 40 min. Raman spectroscopy was utilized to evaluate the quality of the synthesized graphene, which was confirmed to be monolayer with negligible defects , Alfa Aesar (China) Chemical Co., Ltd) with a low-pressure chemical vapor deposition (CVD) method43 before ion irradiation experiments. The TEM grid with the carbon side was placed onto the graphene grown on Cu foil, which was carefully pressed to make the graphene tightly contact the TEM grid. Two drops of IPA were gently dropped on the sample to further increase the adhesion between the graphene and TEM grid after the IPA evaporated. Subsequently, 1 M ammonium persulfate aqueous solutions were used to etch the Cu foil for approximately 50 min. The graphene on TEM grid was then rinsed in deionized water for 5 min by three times to remove residual etchants. After the cleanout, it was dried under an infrared baking lamp at temperature of ~60 °C for 10 min.For characterizing graphene subnanopores, the CVD grown graphene was transferred onto a Quantifoil TEM grid  using a similar direct transfer procedure reported in our previous work78 before ion irradiation experiments. Polymethyl methacrylate  was spincoated onto the graphene grown on Cu foil with the first rotate speed of 500 rpm for 20 s and then 3000 rpm for 40 s. The sample was then placed on a heating plate  at temperature of 150 °C for 5 min to cure the PMMA layer. Similar to the transfer procedure of the graphene on TEM grid, 1 M ammonium persulfate aqueous solutions were used to etch the Cu foil for about 50 min, and the sample was rinsed in deionized water for 5 min by three times to remove residual etchants. Subsequently, the PMMA/graphene composite was transferred onto the prepared PET membrane, and it was dried in ambient conditions for 30 min followed by desiccation under an infrared baking lamp at temperature of ~60 °C for 30 min. Trichloromethane was used to dissolve the PMMA of the sample for 18 h, which was then rinsed in IPA for 5 min to remove residuals. At last, the graphene supported on PET membrane was dried under an infrared baking lamp at temperature of ~60 °C for 30 min.For measuring ion currents of the GPETNC, the CVD grown graphene was transferred onto a prepared PET membrane with a conically shaped nanochannel using a similar wet transfer procedure reported in our previous works13 cm−2 (with current of ∼30 nA) at room temperature and pressure of ~2.2 × 10−6 Torr using the 2 × 1.7 MV electrostatic accelerator of State Key Laboratory of Nuclear Physics and Technology, Peking University, China. To create a nanometric track (narrow damage trail) in PET membrane for subsequent track-etching procedure, a pristine PET membrane  was perpendicularly irradiated by a single 86Kr26+ ion with energy of 2.15 GeV at room temperature using the sing-ion-hit system at the Lanzhou Interdisciplinary Heavy Ion Microbeam of Heavy Ion Research Facility in Lanzhou, Institute of Modern Physics, Chinese Academy of Sciences, China.To introduce subnanopores in graphene, the transferred graphene on TEM grid or PET membrane was perpendicularly irradiated by Au ions with energy of 500 keV and fluence of 1 × 1040. The prepared PET membrane with a nanometric track irradiated by a single swift heavy ion was chemically etched in a custom-designed system with one conductivity cell containing the etchant of 2 M NaOH and the other of the stop medium of 1 M HCOOH as well as 1 M KCl, which was heating in a water bath at temperature of 60 °C. A Keithley picoammeter  using gold electrodes was applied to monitor the ion current through the PET membrane in the etching procedure under the applied voltage of 1 V at the side of the etchant. When the ion current reached ~0.5 nA in ~10 min, which indicated the birth of a conical nanochannel, the etchant was replaced by the stop medium to terminate the etching process, and the sample was subsequently rinsed in deionized water to remove residuals.The PET membrane with a single conical nanochannel was fabricated by the asymmetric track-etching technique using a similar procedure reported in our previous works82 was utilized to reduce surface contaminations of graphene after ion irradiation experiments. Nanoporous graphene on TEM grid was embedded in activated carbon powders in a glass vial and heated on a heating plate . The temperature of the sample was increased from room temperature to 200 °C with rate of 2.5 °C/min, and this temperature was held for 30 min. The sample was subsequently let to cool down to room temperature and carefully taken out of the glass vial, followed by being gently purged with a nitrogen stream to remove the residual activated carbon powders.A dry-cleaning method with activated carbon powders−5 Torr for at least 12 h to further reduce surface contaminations, allowed to cool down to about 60 °C under the vacuum, and then immediately loaded into the microscope column. Room temperature HAADF-STEM imaging was performed on the Nion U-HERMES200 aberration-corrected STEM located in the Electron Microscopy Laboratory of Peking University. The microscope was operated at an accelerating voltage of 60 kV to avoid electron radiation induced damage to the graphene while imaging85. The convergence semi-angel was 35 mrad with an annular recording range of 80-210 mrad. The electron probe diameters were focused to ~60 pm, and the beam current was measured as 28 pA for the imaging. The STEM images were recorded with 4096 × 4096 pixels, and each pixel collection time was 128 ms. The images were filtered using a Gaussian function implemented in the microscope software, and the s-curves of the images were adjusted to increase the contrast between carbon atoms and subnanopores.Before imaging, the dry-cleaned graphene on TEM grid was baked at temperature of 160 °C and pressure of <10I–V curves were measured by a Keithley picoammeter  using Ag/AgCl electrodes in a custom-designed system with two conductivity cells filled with chloride salt solutions at the same concentration or one cell filled with 1 M and the other with 1 mM KCl solutions for measuring cation/anion selectivity. Voltage was applied at the base side of the sample  selectivity is identified using the Goldman–Hodgkin–Katz (GHK) model51, which does not explicitly rely on the channel size and has been successfully utilized to characterize the ion selectivity of graphene nanopores43. Briefly, the K+/Cl− selectivity SGHK is calculated according to the GHK voltage equationkB is the Boltzmann constant, T is the temperature, e is the elementary charge, chigh and clow are the concentrations of KCl solutions at the two sides of the sample, and Vrev is the reversal potential, which is the applied potential where the net current is zero. The Vrev of the GPETNC was estimated to be 254 mV by cubic smoothing spline fitting of the measured I–V curve at chigh = 1 M and clow = 1 mM  is defined43 asGi is the measured channel conductance in the cation-chloride solution at different voltages, Qi is the electric charge of cation i, and μi is the electric mobility of cation i in bulk solutions. Hence, the inter-cation selectivity Si of cation i (relative to that of K+) satisfies43Inter-cation selectivity is obtained by directly comparing the ion conductance in different chloride salt solutions at the same concentration, which is effective to describe the ability of ion channels to discriminate different cations under the circumstance that ion current due to Clψ, F, ε, R, and T are respectively the electric potential, Faraday constant, dielectric constant of the medium, ideal gas constant, temperature, and zi, ci, Ji, Di are respectively the electric charge, concentration, flux, and diffusion coefficient of ion i. The region to be solved and corresponding boundary conditions are shown in the schematic diagram of the model of GPETNC in Supplementary Fig. Ion transport in nanochannels is numerically calculated by solving the Poisson–Nernst–Planck (PNP) equations (Eqs. –6)) usi) usi6)) The theoretical model based on numerically solving the PNP equations described above is powerful to study the transport of charged species in ion channels. It is worth noting that there exist a few limitations for such a model, which include, for example, the neglect of steric hindrance caused by the finite volume of ions, dielectric effects such as partial dehydration, and weak van der Waals forces. However, those neglected effects can only quantitatively affect the calculated results, such as the values of ion concentration and electric potential, but they do not influence the correctness of the conclusions, because the electrostatic interaction and ion diffusion are nicely considered in the model.Supplementary InformationPeer Review File