Renewable energy sources are more advantageous than fossil fuels because they are unlimited in quantity, do not cause great harm to the environment, are safe, and create economic value by reducing foreign dependency because they are obtained from natural resources. With nanotechnology, which enables the development of different technologies to meet energy needs, low-cost and environmentally friendly systems with high energy conversion efficiency are developed. Renewable energy production studies have focused on the development of hydrovoltaic technologies, in which electrical energy is produced by making use of the evaporation of natural water, which is the most abundant in the world. By using nanomaterials such as graphene, carbon nanoparticles, carbon nanotubes, and conductive polymers, hydrovoltaic technology provides systems with high energy conversion performance and low cost, which can directly convert the thermal energy resulting from the evaporation of water into electrical energy. The effect of the presence of water on the generation of energy via the interactions between the ion(s) and the liquid–solid surface can be enlightened by the mechanism of the hydovoltaic effect. Here, we simply try to get some tricky information underlying the hydrovoltaic effect by using DFT/B3LYP/6-311G computations. Namely, the physicochemical and electronic properties of the graphene surface with a water molecule were investigated, and how/how much these quantities (or parameters) changed in case of the water molecule contained an equal number of charges were analyzed. In these computations, an excess of both positive charge and negative charge, and also a neutral environment was considered by using the Na The excess energy in the water, which can flow and evaporate spontaneously by adsorbing thermal energy without being affected by environmental conditions, is separated from the surface in the form of latent heat3. The evaporation potential and current, which continues as long as the evaporation of water continues, provide the conversion of thermal energy into electrical energy4. Due to the unique characteristics of both the water molecule and graphene surface in terms of the structural, electronic, and thermal properties, water-graphene systems have been increasingly studied in many areas7. The graphene structure consists of the sp2 hybridized carbon atoms and thus the electron delocalization on the surface plane is responsible for the in-plane heat conductivity, while out-of-plane thermal conductivity can be provided by the weak dispersion forces or van der Waals interactions8. Also, the water molecule has been reported with a vital role in the harvesting of new-generation energy such as kinetic energy by hydrovoltaic tools, gradient, and evaporation energy9.Natural water is the most abundant clean and sustainable resource on earth, covering approximately 71% of the earth's surface and capturing approximately 70% of the solar energy received by the earth's surface9. Electric output generation can be improved using porous materials such as carbon black sheets and nanowires with nano-sized channels11. In this direction, studies have focused on finding the amount of energy the system has due to the effect on the water drop and graphene surface.Hydrovoltaic systems, which produce electricity from the direct interaction of water with the material, are preferred instead of existing technologies used to obtain electrical energy from the kinetic energy of water. Energy from water is converted into electrical energy by hydrovoltaic effects provided by mechanisms such as flow potential, fluctuation potential, shrinkage potential and evaporation-induced electricity, and gradient-induced ion diffusion. When solids come into contact with water, ions with opposite charges to the surface charge are attracted and an electrical double layer (EDL) is formed at the water–solid interface. EDL-based electrokinetic effects, such as flow potential, can convert the kinetic energy of water flowing through narrow channels into electrical energy. However, the formation of a high flow potential is possible with a large pressure gradient. By placing a single layer of graphene or carbon on the film formed along the surface of the rippling water, wave energy is collected and the wave potential is converted into electrical energy. A drop of water is dripped onto a graphene sheet to create an attractive potential and is used to collect the kinetic energy of raindrops and solar cells operating on rainy days. Evaporation of water in our environment always takes place as a normal process, during which a large amount of thermal energy is converted into latent water vapor energy. Sustainable tension can be obtained from the evaporation of natural water using a small porous piece of carbon black film partially immersed in water. Any energy gradient is equivalent to a repulsive force that can do work on the system, such as moving charge carriers. Osmotic energy is the most common gradient energy and is derived from the directional diffusion of ions along a concentration gradient in water. Low ion concentration and the large distance between ions and solid surface limit the interaction between water vapor and solid surface, resulting in low electrical output12. The hydrophilic and hydrophobic phenomena have great importance in predicting/evaluating the interactions that exist in a specific adsorbent (a solid surface)-adsorbed (water)15. In this respect, Jiang et al. explored the catalytic effect of a perpendicular electric field on the reversible transition of graphene with water from hydrophobic to hydrophilic by using first-principles calculations; they disclosed that the electric field could act as a switch to reversibly change the graphene from hydrophobic to hydrophilic in the presence of water vapor16. On the other hand, polarizability holds a crucial position in the modulation of its hydrophilicity-hydrophobicity dynamics, establishing the groundwork for its interactional paradigm with water molecules. Thus, this intrinsic characteristic significantly influences graphene's physical properties, thus broadening the potential for its applicability across a variety of scientific domains. In this regard, a comprehensive analysis of graphene's electronic polarizability had been conducted using multiple theoretical methodologies, including Kubo's linear response formalism for frequency and wave-vector-dependent polarizability within the random phase approximation17. Furthermore, the first-principles density functional approach was used to analyze the properties of graphene multilayers and graphite under an applied electric field; the specific regions of the designed graphene multilayers were explored in terms of the induced charge under applied electric fields18. In a study, an analytical model, in which parameters such as polarizability and bending stiffness were derived from numerical fits to simulation data was introduced to predict the alignment of infinitely wide Graphene Nanoribbons (GNRs) to an electrostatic field; the model revealed that there was a linear correlation between alignment angle, field strength square, and graphene length for small deformations19. Recently, Escalona et al.20 examined the water-graphene interface in distinct systems, one with graphene in water and another with a water droplet on graphene, under an electric field; their MD results implied that water's orientation near graphene was affected by graphene's polarizability, particularly under an electric field.Recently, molecular dynamic computations were performed to explore the effect of the water humidity on the graphitic surface and suggested that the graphene surface is quite wettable and presents hydrophobic behavior in case it was covered by the double water layers21. In the past, Monte Carlo simulations were conducted to elucidate the water adsorption on the graphene surface at various temperatures at 313, 316, and 323 K, and the results showed that water molecules would prefer to merge at the edges of the graphene surface rather than adsorb onto the basal graphene plane22. Moreover, an ab initio study is reported to investigate how many electrons each ion can attract from the polymer (PET)-enclosed graphene surface and whether it is suitable for harvesting water energy23. Accordingly, the authors suggested that the shortened surface thickness or more substrate dipole can boost the output power23. In a remarkable, Yin et all demonstrated that the electrokinetic potential can be produced in a graphene sheet that moves between air and solution interfaces, and the results of that work have quite promised to design self-powered sensors and monitors24. In designing smart materials, especially, graphene-based systems are getting great attention because of promising structural relevant properties such as mechanical and thermal features, and applicable surface area25.In the case of the adsorption of the water molecule issue has a complexity because of the possible H-bonding between the surface and water molecule or between the water molecules. Hence, it is hard to evaluate this kind of interaction just only using a limited factor such as H-bonding tendency, surface properties, hydrophilicity, etc.One of the main issues in the contemporary age, even the most important, is to generate energy from ergonomic, economical, renewable, etc., then for a green future. Nowadays, waste energy is also processed to re-generate energy, but research has shifted towards nanogenerators due to these (secondary) energies obtained being very low efficiency. Although the three main mechanisms of the hydovoltaic nanogenerators, as a class of nanogenerators, have been known, the water effect on the solid surface (electrode) has not been explained and keep stand as controversial. In this respect, the reported data in the literature are generally related to developing new electrodes that can harvest hydrovoltaic energy, but not to deeper reasons for them, especially electronic structures and relevant electronic properties.17. These clusters subsequently expand and coalesce at the peripheries of the graphene layers, instead of adsorbing onto the graphene's basal planes. This phenomenon can be attributed to the stronger electrostatic interactions, particularly hydrogen bonding, between water molecules, which supersede the relatively weaker dispersion interactions with the basal planes of graphene17.The finite-dimensional constraints of graphene have noteworthy implications on the manifestation of the hydrovoltaic effect, an outcome that diverges markedly when considered in the context of a hypothetically infinitely extended graphene plane. The existence of boundary effects and the limited expanse in these finite graphene constructs may notably modulate the interactions and spatial distribution of ions and water molecules on the graphene interface, leading to consequential influences on hydrovoltaic energy generation. As reported, the simulation outcomes demonstrate that water exhibits a preference for adsorption around the functional groups of graphene, fostering the formation of clustersWhile the theoretical exploration of infinite graphene planes presents an intellectually engaging discourse, it is essential to acknowledge that the preponderance of graphene's practical implementations is based on finite-sized components. Consequently, despite the intrinsic limitations inherent to the finite nature of these graphene-based systems, the insights garnered from such studies continue to offer substantial value and applicability across multiple domains.30 such as doped by metal or transition metal, nanostructures, or graphene quantum dots, etc. For instance, the Density Functional Theory (DFT) based on first principles examined the impact of different impurities on the structure, electronics, and charge transfer in graphene to explore the possible usage for graphene in optoelectronics and other devices requiring switching capabilities31. Other first-principles DFT computations were reported to explore the gas-sensing performance of transition metal-doped graphene systems32. Furthermore, Kumar et al. employed DFT computations to examine the impact of doping graphene with platinum group elements (PGE); they calculated the energy gap, UV–vis absorption spectra, and Mulliken population to explore the potential uses in wearable electronics, optoelectronics, sensors, and solar cells33. Furthermore, the graphene oxide nanosheet (GON) was modified based on the Lerf-Klinowski model and investigated the impact of integrating Be, B, N, O, and F atoms on GON's properties by using the B3LYP-D3/6–31 + G34. Also, the DFT calculations and MD simulations were utilized to investigate boron-modified graphane nanoparticles to determine the reactivity and hydrogen binding potencies to examine their optoelectronic properties, indicating potential applications in clean energy and materials science35. In another study on research of BX  doped twin-graphene, the authors utilized the DFT based on first-principle calculations to explore the possible potentials of the designed systems, which would produce clean energy36.Until now, DFT computations in terms of molecular mechanics and dynamic perspective have been applied successfully to graphene/ graphite systemsTo understand the basic mechanism of the surface-based hydrovoltaic effect; it is necessary to understand the interactions and energy relations of water, ions, and electrons on the solid–liquid surface. In this direction, in our study, the physicochemical effects of a water molecule on the graphene surface as a typical model were investigated. In addition, it also analyzed how the physicochemical parameters change when the water molecule contains an equal number of charges, excess positive charge or excess negative charge. Our work serves as a significant step toward understanding the intricate dynamics at the solid–liquid interface, crucial for optimizing the hydrovoltaic effect in various applications. By providing detailed insights into the physicochemical interactions between the ions and water and finite graphene, we hope that the results of this work pave the way for future research aimed at enhancing the efficiency and performance of graphene-based hydrovoltaic devices.37 package and analyses and representations of the results were made by using GaussView 6.0.1638. At first, the electronic structures of isolated molecules, ions, and salt were confirmed by the absence of no imaginary frequency following the geometry optimization by the default convergence criteria40, at B3LYP/6-311G** level42 and basis set45.All electronic structure computations were performed by G0948. As known well, thermodynamic and thermochemical properties for the relevant systems are calculated by using the total partition function which depends on the freedom degrees of the translational (Qtrans.), rotational (Qrot.), vibrational (Qvib.), and electronic (Qelec.) movements47. For a general system, the molecular partition function is defined asThe thermochemical and physical characteristics of all studied species were determined by the quantum statistical principles49.The vibrational partition function for the asymmetric top molecules is given below, and the degrees of vibrational freedom contribution to the thermochemical quantities can be calculated by Eqs. –5)46–4946–495)46, Evib. “vibrational thermal energy”, Svib. “vibrational entropy”, and Cvvib. “vibrational heat capacity” is defined as29. The terms are expressed as νj → ” jth fundamental frequency”.HereI “ionization energy” and A “electron affinity”50 are used for determining the reactivity properties by using the following equations.Furthermore, the values max → “the maximum charge transfers capability index”56, ω- “the electrodonating power” and ω + “the electroaccepting power”57, and ΔEback-donat. “back-donation energy”58.Here, χ → “electronic chemical potential” η → “global hardness”, ω → “electrophilicity”, ΔNThe confirmed structures of the possible interactions between Graphene and anionic, cationic, salt, and water were presented in Fig. G (isolated graphene) and S1 (with water on the Graphene surface) were calculated as 1.42 Å whereas this bond for S3 and S4 was determined as 1.43 Å. In the past, the C–C length for the graphene unit was determined in the range of 1.34–1.49 Å62 depending on the type of the doped atom. Namely, the C–C bond length for the Mg-doped graphene core was calculated as 1.34–1.44 Å 31, whereas for the O-doped Graphene, the nearest C–C length was predicted as 1.49 Å 33. On the other hand, this length for the isolated graphene core was mostly determined as 1.42–1.43 Å 59. Also, the neighbor C–C bond lengths for the B, N, O, and F-doped graphene structures were estimated at 1.403, 1.416, 1.485, and 1.353 A, respectively63. Also, the O–H–C1 length for all systems was calculated as 2.50, 2.23, 2.29, and 5.57 Å: the oxygen distance to the graphene surface did grow longer with the presence of the salt (NaCl) while it did narrow with the existence of the ions (Na+ or Cl−). The geometric parameters show that the electronic attraction force between the Na+ ion and the graphene surface is larger than the other systems, and therefore the Na+ ion (S3) is placed parallel to the graphene surface. On the other hand, the distance between the Cl- ion and the graphene surface increased due to the electronic repulsion forces between the charge density on the graphene surface and the charge cloud of the Cl- ion, and Cl–– H-C1 length was calculated as 2.61 Å. Also, Na+–C3 and Cl––C3 lengths were calculated at 4.38 and 4.10 Å, respectively, whereas the Na+–C2 and Cl––C2 distances were calculated at 2.71 and 4.78 Å. The C1-C2 –Na + and C8–C4–Na angles for S3, which the placement of the Na + ion on the graphene-like layer, were calculated as 120.9° and 75.7°, respectively. Moreover, the C4–C2–O angle for S1 was calculated as 179.0° which implied that the water molecule was positioned approximately at the same plane with the graphene structure, whereas this angle for S4 was calculated as 72.1° which was a sign that the water molecule  was placed on the surface at an angle. In addition, the C2–C5–O angle for systems S1–S4 was calculated at 99.5°, 108.2°, 98.3°, and 61.7°, respectively. From Table sp2 hybridization.From Table S2 and S4 were calculated higher than those of the other systems because of the existence of the electronegative chlorine atom. Namely, the order of the dipole moments of G and S1-S4 (in D unit) was determined as 22.172 (S2) > 10.172 (S4) > 9.727 (S3) > 2.358 (S1) > 0.000 (G), whereas the polarizability values of them (in au) were determined as 308.940 (S4) > 308.878 (S2) > 291.786 (S3) > 288.305 (S1) > 279.745 (G). In the context of molecular electronic properties, our findings underscore a notable variability among the studied systems. Specifically, state S2 demonstrates the most pronounced dipole moment, standing at 22.172 D. This places S2 as the most polar entity within the studied set, implying a substantial charge separation within the molecule64. Contrastingly, state S4 exhibits the highest polarizability, with a measure of 308.940 atomic units. This suggests a heightened response of the electron cloud to perturbations in the external electric field, indicative of a more deformable electronic distribution under the influence of an external field67. Remarkably, ground state G is characterized by the least values for both dipole moment and polarizability, revealing its lower polarity and relative electron cloud rigidness. This set of observations facilitates a deeper understanding of the electronic characteristics of the various states, potentially driving further investigations into their interactive behaviors and reactivity profiles.Also, the calculated physical and thermochemical properties were summarized in Table S4) > 202.809 (S3) > 202.594 (S2) > 200.998 (S1) > 188.657 (G); highly portion of it came from the vibrational motions. Also, heat capacity and vibrational heat capacity  orders were calculated as of 85.957 (S4) > 81.914 (S3) > 80.106 (S2) > 75.865 (S1) > 64.619 (G) and 79.996 (S4) > 75.952 (S3) > 74.144 (S2) > 69.903 (S1) > 58.658 (G), respectively. Furthermore, the translational entropy values of the designed adsorbent-absorbance systems (S1-S4) were calculated at 43.167 (S1), 43.478 (S2), 43.375 (S3), and 43.666 cal/mol K (S4), whereas the rotational contribution to the entropy was determined at 34.764 (S1), 35.645 (S2), 34.938 (S3), and 35.525 cal/mol K (S4). As can be expected, the greatest contribution to the entropy was sourced from the vibrational freedom degree of all systems and calculated as 38.537 (G), 61.221 (S1), 69.230 (S2), 68.966 (S3), and 87.074 cal/mol K (S4).Thermal energy values  changed in the order of 204.610  were presented in Table S3 process is greater than in the others. Namely, the enthalpy change (in kJ/mol) for the assumed systems was calculated as S1 (−12.08) < S2 (−113.94) < S4 (−119.03) < S3 (−176.71); the presence of the cation Na+ made the enthalpy change of lowered. Furthermore, the adsorption-free energy changes indicated that the adsorption processes would occur spontaneously for the designed systems S2–S4. From Table S2–S4 were determined as S2 (−52.86) < S4 (−57.45) < S3 (−115.86), while the free energy changing for S1 was determined as 14.84 kJ/mol, which indicated the adsorption system could not occur spontaneously.The adsorption energies of the designed systems were calculated by using the following equations.entclass1pt{minimaS1–S4 were given in Table + cation can be said that makes the graphene more reactive to the outer system(s) with the ΔEgap value of 3.794 eV that was greater than those of the Cl- and NaCl salt. As can be expected, the Na+ ion (1s22s22p6), with the electron deficiency, made the graphene more electrophile, while the water molecule made it less electrophile. From Table G and systems S1–S4 were calculated in the order of S2 (0.040) < S1 (0.117) < G (0.125) < S4 (0.175) < S3 (0.441). Also, the calculated Δεback-donat. values revealed that the water molecule made G more stable via back donation whereas the Cl- made it less stable. The order of Δεback-donat. values (in eV) for systems S1–S4 was calculated as -0.501 (S1) < −0.474 (S3) < −0.452 (S4) < −0.199 (S2). In Table 2O, Cl-, Na, and NaCl species were given for the prediction/direction of the possible electronic movement between the surface and each of the chemical species. Here the frontier molecular orbital densities for the isolated molecules and designed systems were visualized in Figs. S2–S4 expanded on the Cl- ion and which was a pictorial proof of the electronic movement from the HOMO of Cl- ions to the LUMO of graphene.The possible reactivity identifiers of the isolated molecules and designed systems a+ ion 1s2s22p6, w+) were calculated as 1.405 eV (without water) > 1.279 eV (with water). On the other hand, for the electrically neutral environment , it can be said that the electronic movement from the HOMO of Graphene to the LUMO of NaCl is more likely than the opposite version. The energy ranges for these two possible electronic transition scenarios were calculated as follows: ΔE2 (3.561 eV) < ΔE1 (4.635 eV).To predict the direction of possible electron, transfer between the graphene surface and the considered ions and salts, the energy ranges for each designed system were calculated using the equations below, and the results are given in Table Without water,With waterS2–S4 were determined as S2 (−52.86) < S4 (−57.45) < S3 (−115.86), which indicated the adsorption process could occur spontaneously. Also, the energy range values strongly implied that the charge transfer could be towards from the HOMO of the S1 to LUMO of the Na+ due to the order of it as ΔE2 (1.279 eV) < ΔE1 (37.833 eV). The results showed that the positively charged Na+ ion makes the Graphene-water system have higher energy and thus it is time to be focused on the positively charged G-water systems to produce the next-generation energy.In this study, we attempted to evaluate the presence of water on a solid surface about how higher hydrovoltaic energy can be obtained with which charged ion. Here, the computational results indicate that hydrovoltaic nanogenerators with higher voltage output can be produced on cation-rich water-included surfaces. The adsorption-free energies of systems


Removal of trace organic chemicals (TOrCs) in aquatic environments has been intensively studied. Some members of natural microbial communities play a vital role in transforming chemical contaminants, however, complex microbial interactions impede us from gaining adequate understanding of TOrC biotransformation mechanisms. To simplify, in this study, we propose a strategy of establishing reduced-richness model communities capable of removing diverse TOrCs via pre-adaptation and dilution-to-extinction.Microbial communities were adapted from tap water, soil, sand, sediment deep and sediment surface to changing concentrations of 27 TOrCs mixture. After adaptation, the communities were further diluted to reduce diversity into 96 deep well plates for high-throughput cultivation. After characterizing microbial structure and TOrC removal performance, thirty taxonomically non-redundant model communities with different removal abilities were obtained. The pre-adaptation process was found to reduce the microbial richness but to increase the evenness and phylogenetic diversity of resulting model communities. Moreover, phylogenetic diversity showed a positive effect on the number of TOrCs that can be transformed simultaneously. Pre-adaptation also improved the overall TOrC removal rates, which was found to be positively correlated with the growth rates of model communities.This is the first study that investigated a wide range of TOrC biotransformation based on different model communities derived from varying natural microbial systems. This study provides a standardized workflow of establishing model communities for different metabolic purposes with changeable inoculum and substrates. The obtained model communities can be further used to find the driving agents of TOrC biotransformation at the enzyme/gene level.The online version contains supplementary material available at 10.1186/s12934-023-02252-6. In recent years, the ubiquitous and frequent detection of trace organic chemicals (TOrCs) in aquatic environments is of increasing concern . DespiteIn the last decades, cultivation-independent methodology using next generation sequencing has been developed to explore whole microbial systems –11. HoweAcinetobacter strain and Bacillus strain in a synergistic consortium resulted in higher degradation efficiency of herbicide bromoxynil octanoate than either strain individually [According to current studies, model communities can be assigned to the categories of synthetic, semi-natural and natural, and they might consist of two-species or higher member complex co-cultures, and enriched model systems . Model cvidually .In this study, we aim to establish new model communities that can be applied to mechanistically investigate biological TOrC transformation processes and to help improve biotransformation technologies. A combination of enrichment steps and the dilution-to-extinction method were tested in order to provide a robust, standardized protocol for obtaining diverse model communities that can biotransform TOrCs. We hypothesized that (i) the pre-adaptation process will benefit the selection of model communities in terms of diversity, and (ii) the phylogenetic diversity of the resulting model communities will influence the TOrC removal rates. We followed a series of enrichment steps to adapt the inoculum to TOrC environment and subsequently used the dilution-to-extinction approach to establish thousands of potential model communities. We also tested the effects of the inoculum and its initial diversity on the protocol in order to provide a universal framework for several starting communities for TOrC biotransformation research.A broad range of TOrCs with different properties and uses are present in WWTP effluents . To adap2HPO4·2H2O, 746 mg KH2PO4, 70 mg MgSO4, 120 mg (NH4)2SO4, 1 mg Ca(NO3)2, 0.1 mg H3BO3, 2.5 mg FeSO4·7H2O, 0.75 mg MnSO4·H2O, 1.3 mg ZnSO4·7H2O, 0.25 mg CuSO4·5H2O, 0.3 mg Co(NO3)2·6H2O, 0.15 mg Na2MoO4·2H2O, 0.01 mg NiSO4·7H2O) [27 TOrCs were prepared as a mixed stock solution at the concentration of 50 μmol/L per compound. Samples for inoculation were obtained from tap water , technical sand treated with tertiary effluent , soil  and sediment  on November 2021. Samples were immediately taken to the lab for storage at 4 °C until usage. Sediment was collected by a gravity corer and was divided as sediment surface (0 m) and sediment deep (0.3 m). Growth medium was prepared with mineral salts (1L: 448 mg NaO4·7H2O)  and addi–6, making it possible to determine the removal of TOrCs as the only carbon source in P6. In the final step P6, the aim was to get close-to-natural conditions of TOrC concentrations, as an adjustment to the final concentration of the degradation assays. The pre-adapted microbial communities from P6 were used as the inocula for the dilution-to-extinction steps setting up the model communities.Each sample was used as inoculum  and was directly added into 450 mL growth medium in a 1 L sterile glass bottle in triplicates, resulting in 15 bottles in total. They were incubated in stationary in the dark at room temperature for one month. Afterwards, 450 mL suspension was discarded and replaced by fresh growth medium. The pre-adaptation process consisted of six phases which took six months. TOrC concentrations were adjusted in each phase . This process was designed as a selective enrichment to let microbial communities adapt increasingly to TOrCs (the sixth phase with 50 nmol/L for recovery) and to decreasing alternative sources of carbon. Furthermore, to avoid dramatic environmental perturbations, TOrC concentrations increased gradually from 50 to maximum 2500 nmol/L. Despite the increasing selection pressure brought by TOrCs, the variation of concentrations was still in the range of real-world conditions  for 10–15 min at room temperature in the dark . Samples6 cells/mL (the ubiquitous density observed in aquatic environment) [Microbial communities from adapted  or non-adapted  inoculum were serially diluted to 0, 0.1, 1, 10, 50, 100, 200, 500, 800, 1000, 2500, 5000, 10,000 cells/mL using growth medium spiked with 5 nmol/L TOrCs mixture in 96 deep well plates to establish model communities. The P6 inocula were only diluted from 0 to 1000 cells/mL, since we expected a more constant initial diversity. 48 wells of a 96 deep well plate were inoculated with 1 mL suspension from each dilution, resulting in 4704 wells from all dilutions and all inocula, and 16 blank wells (without cells) as control. All plates were incubated in the dark at room temperature for 21 days, which was shown to reach the stable growth phase according to our pre-experiment. After 21 days, cell counting was used to screen the wells for positive growth defined as a minimum of 1 × 10ronment) , 33.Microbial communities with different diversity were diluted to 1000 cells/mL, and then 1 mL suspension was inoculated in a new plate spiked with 5 nmol/L TOrCs to investigate their removal rates  within 21 days. Similarly, all P6 inocula and blanks (TOrC medium without cells and with autoclaved cells) were incubated in the plates for the same period. Blanks acted as controls to evaluate the abiotic TOrC degradation. Cell counts were monitored at 2, 5, 7, 8, 12, 14, 16, 19, 21 days to estimate the growth rates and final cell counts. TOrC concentrations were measured at the start (0 d) and end (21 d) time point by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) following Müller et al. . BrieflyPREP Plus beads  and AMPure XP beads  according to the manufacturer's instructions. Library preparation was performed using native barcoding kit 96  following the manufacturer's instructions. A total of 75 μL library pool was loaded into an Oxford Nanopore R10.4 flow cell and sequenced with MinION™ Mk1C device. The run was stopped once desired number of reads  were achieved.Samples of the initial inocula P0, adapted inocula P6 and final model communities were subjected to DNA extraction by using the DNeasy PowerSoil Pro Kit (QIAGEN) according to manufacturers’ instruction. Extracted DNA was quantified using dsDNA Broad Range Assay  in a fluorometer . 16S rRNA gene was amplified using the 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACTT) primers. The PCR mix included 25 μL 2 × GoTaq® colorless master mix (Promega), 1 μL of each primer (20 μM), 1 μL DNA template, and 23 μL nuclease-free water. Thermocycling conditions included an initial denaturation at 95 °C for 2 min, 30 cycles of denaturation for 1 min, annealing at 56 °C for 1 min and extension at 72 °C for 2 min, and a final extension for 10 min. The PCR products were checked for quality by a 1.5% agarose gel electrophoresis and purified using MagSi-NGShttps://timkahlke.github.io/LongRead_tutorials/BS_G.html). Afterwards we processed the amplicons following Cuscó et al. [https://github.com/rrwick/Porechop). Sequences were then trimmed using NanoFilt v2.8.0 with the parameters of -q 9 and -l 1000 [Raw fast5 reads generated by MinION were basecalled with high accuracy model and converted to FASTQ files using Guppy v3.6.0  analysis was performed based on generalized unifrac distance with alpha setting of 0.5 by R package GuniFrac v1.7 [Filtered OTU table was imported into R v4.2.1 for alpha diversity calculation and phylogenetic diversity comparison. Reads were rarefied to even depth for calculating Shannon index, Simpson index and species richness by R package vegan v2.6.4  and phylrac v1.7 . SignifiWe established model communities that can grow on TOrCs by standardizing a workflow Fig.  that can6 cells/mL) after the first 21 days incubation. The median cell counts of all diluted communities after 21 days incubation in stage 2 point to a putative sigmoid-like function that indicates an inoculum type specific threshold  . In the pre-adapted group, species distributed more evenly  while maintaining some of the taxa that were also dominant in the model communities from the non-adapted inocula . Some rare taxa e.g., Caulobacteraceae and Micrococcaceae occupying less than 2% in all inocula were abundant in the pre- and non-adapted model communities .A closer look at the microbial community structure of the 95 microbial communities revealed that communities from pre- and non-adaptation groups were differently structured according to Unifrac distance  Fig. c. MicrobWe hypothesized that exposing whole communities to TOrCs allows the microbial community to go through a succession. In this succession, naturally abundant but vulnerable to TOrCs microorganisms reduce in numbers while allowing rarer microorganisms to slowly increase in numbers accompanied by members of the community that could act as cornerstones of resilience . Hence, pre-adaptation resulted in an overall reduced richness of the whole community while having a positive effect on the species that can grow on or survive TOrCs and could then thrive in the model communities.t-test, p = 0.16). There were more TOrCs degraded after pre-adaptation (n = 17), including some persistent compounds such as carbamazepine (46.5%) and gabapentin (25.2%), which were removed below 20% in the communities from the non-adaptation group (n = 10) , but to different degrees  regarding their TOrC removal patterns. We hypothesized that phylogenetic diversity of model communities will influence TOrC removal rates, which could explain the dissimilarity between groups. However, the envfit analysis showed that the variation was not correlated with phylogenetic diversity . We also tested the correlation with other variables, i.e., Shannon diversity, estimated growth rate, final cell counts (cell counts at d21), 27 TOrCs average removal rate and removal diversity . We found that only average removal rate  and estimated growth rate  exhibited significant correlation with the variance, indicating the positive effect of model communities’ growth rates on the overall TOrC removal performance. Although phylogenetic diversity was not related to TOrC removal rates, it was found to have a positive correlation with removed TOrC numbers .The relationships between 17 TOrC removal performance and potential microbial traits were investigated by principal coordinate analysis (PCoA) across microbial communities in the pre- and non-adaptation groups. The two main axes explained 41.4% and 16.8% of the variance, respectively  natural communities as Bengtsson-Palme  suggestelum Fig. , whereasNocardiaceae developed from rarity to dominance after pre-adaptation  in the (a) pre-adaptation and (b) non-adaptation group after 21 days incubation, n = 48. Figure S3. Heatmap illustrating 27 TOrCs removal efficiencies by thirty microbial communities. The color legend represents the removal percentage. Figure S4. Taxonomic composition of 11 model communities selected after TOrC removal performance assessment at the genus level. Numbers in the pie chart represent the OTUs belonging to each genus. Figure S5. Comparison of microbial structure between pre- and non-adapted inocula at the family level. Figure S6. Thirty model communities’ frequency on simultaneously removed TOrC number. The removal cutoff is 20%.Additional file 3. OTU tables and metadata: otu_tab_95_model_communities.csv, otu_tab_inocula.csv, metadata.csv.


Sarcoidosis-associated uveitis, is the predominant ocular sarcoidosis presentation, which affects both adults and children. For adults, international ocular sarcoidosis criteria (IWOS) and sarcoidosis-associated uveitis criteria (SUN) are defined. However, for children they are not yet established internationally. Due to the specificity of pediatric manifestations of sarcoidosis, this task is even more challenging. In children, sarcoidosis is subdivided into Blau syndrome and early-onset sarcoidosis (BS/EOS) affecting younger children (< 5 years) and the one affecting older children with clinical presentation resembling adults. Differential diagnosis, clinical work-up as well as diagnostic criteria should be adapted to each age group. In this article, we review the clinical manifestation of sarcoidosis-associated uveitis in adults and children and the sensitivity and specificity of various ocular sarcoidosis diagnostic modalities, including chest X-ray and CT, FDG PET-CT, gallium-67 scintigraphy, bronchoalveolar lavage fluid, genetic testing for NOD2 mutations and serum biomarkers, such as ACE, lysozyme and IL2R. Sarcoidosis is a rare inflammatory condition of unknown cause that affects both adults and children. Its prevalence in adults ranges from 8.1/100 000 in Caucasians to 17.8/100 000 in African Americans. Notably, the disease is less common in children with a prevalence of 0.22–0.27/100000 (Danish study) [The hallmark of the disease is the presence of non-caseating granulomas in affected tissues. Nevertheless, systemic presentation varies among age groups, which often leads to delayed diagnosis in younger patients. It is of note that sarcoidosis in children < 5 years old does not typically involve lungs but skin, joints, and eyes , 3.Ophthalmic manifestations of sarcoidosis can involve any part of the eye and its adnexa in the inflammatory process . The preConsidering the above, certain patients diagnosed with uveitis e.g. patients with granulomatous or posterior uveitis should undergo sarcoidosis screening as a routine workup. However, a definite diagnosis is often difficult to obtain as according to the revised IWOS Criteria for diagnosis of ocular sarcoidosis a biopsy of the lesion is required . It is fThis clinical scenario is even more complex in children. The lack of a typical pattern of systemic presentation and the absence of standardized guidelines on ocular sarcoidosis in children makes a clinical diagnosis of ocular sarcoidosis difficult .Here, we present overlapping as well as distinguishing features of ocular sarcoidosis in children and adults followed by a differential diagnosis and novel diagnostic approach. The contrast between features of the disease across various age groups aims to increase clinicians' awareness regarding this diagnosis and ensure a timely diagnosis for more patients, particularly those under 5 years of age.We utilized the Pubmed database to search for relevant publications. Manuscripts published between 2013 and 2022 were utilized as the primary source of data. Ocular sarcoidosis, sarcoidosis, early onset sarcoidosis, Blau syndrome, and uveitis served as primary search terms.There are no firmly established guidelines dedicated to the diagnosis of sarcoidosis. However, it is agreed that the disease is deemed highly probable in an individual with typical clinical features, non-caseating granulomas in the histopathological examination, and in whom other granulomatous diseases have been ruled out .The 2014 guidelines issued by the World Association of Sarcoidosis and other Granulomatous Diseases point out that certain clinical manifestations and imaging or laboratory findings are suggestive of the disease and these patients should undergo sarcoidosis screening . ClinicaConsidering that the clinical picture of the disease is non-specific, a biopsy of the lesion is usually endorsed by sarcoidosis experts . HoweverThe course of systemic sarcoidosis in children older than 5 years is similar to the adult disease described above , 18. CliSystemic sarcoidosis usually affects teenagers with a mean age of 13–15 years. Notably, its prevalence is much lower than in adults and a recent Danish study showed that it equals 0.22–0.27/100 000 , 20.Sarcoidosis in children younger than 5 years is usually considered separately and is labeled as Blau Syndrome when there is a family history proving an autosomal dominant trait or Early Onset Sarcoidosis (EOS) when no family history is evident . ChildreNotably, Blau syndrome and EOS are a result of Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), previously known as CARD15 gene mutation . NOD2 se. Other expanded manifestations of BS/EOS are fever, pneumonitis, bronchial granulomas, hepatosplenomegaly, hepatic granulomas, sialadenitis, erythema nodosum, leukocytoclastic vasculitis, transient neuropathies, arterial hypertension, pericarditis, pulmonary embolism, granulomatous glomerular and interstitial nephritis, and chronic renal failure [Data from the Blau syndrome registry shows that patients with Blau syndrome develop rash, arthritis, and uveitis with a median age of 1.1 years, 2 years, and 4.4 years respectively . Further failure . Expande failure . Neurolo failure , 32.Ophthalmic manifestations of sarcoidosis affect virtually any part of the eye and its adnexa. The most common form includes uveitis  and conjunctival granuloma. The former may lead to significant visual disability, whereas the latter is usually asymptomatic. Other ophthalmic manifestations of sarcoidosis consist of dacryoadenitis, orbital inflammation, eyelid granuloma, madarosis, poliosis, keratitis, and optic neuritis .Coulon et al. reported that among 194 adult patients with biopsy-proven and presumed uveitis only 9% had additionally other ocular involvement, including conjunctival node (2%), scleritis (1%), episcleritis (1%), optic neuropathy (5%), dacryoadenitis  .Ocular sarcoidosis might thus globally refer to any inflammation of the eye and its adnexa  and can P = 0.009) and have less granulomatous anterior uveitis  [p < 0.001), respectively 34.3, 43.1 and 57.8 years [Ocular sarcoidosis as well as systemic sarcoidosis show some differences among the ethnic groups. Systemic involvement of sarcoidosis is more frequent in North Africans than in White Europeans, who show a higher frequency of isolated ocular involvement at onset and during follow-up . Anterio< 0.001) . Afro-Ca.8 years .Features of sarcoidosis-associated uveitis are included in numerous cohort studies , 36, 37 Uveitis in adults presents typically as a bilateral and chronic disease. It is predominantly either panuveitis (8–67%) or anterior uveitis (20–52%) and it varies among the studies. Intermediate uveitis is less frequent (3–19%) and isolated posterior uveitis is rare (4–29%) , 38–40.Anterior segment inflammation is granulomatous in 48–60% , 35, 40.Although intermediate uveitis is not frequent, vitreous involvement is often observed with snowballs or string of pearls in 17–50% of patients , 39.The most common manifestation of sarcoid-associated uveitis in the posterior segment is multifocal choroiditis Fig. B followeThe available data concerning children is very limited. In Table The paucity of data allows only for preliminary conclusions. Granulomatous anterior uveitis is present in 23–31% of pediatric patients above 5 years old. One study indicates non-granulomatous anterior uveitis as more prevalent in children than granulomatous anterior uveitis . InflammMulticenter studies on patients with Blau syndrome reported a 76–81% prevalence of ocular involvement  , 31.The typical ocular presentation in Blau syndrome is bilateral panuveitis observed in 43–85% of patients   syndrome .Other causes of granulomatous uveitis may also be included especially in cases of atypical presentations or in immunocompromised patients: toxoplasmosis, lyme, cat scratch disease, cryptococcosis (in immunocompromised patients) or even endophthalmitis .Rarely sarcoid uveitis can have less typical presentations eg. unilateral (16%)  or non-gNon-caseating granuloma remains a histopathological hallmark for sarcoidosis and is included in diagnostic criteria of definite ocular sarcoidosis  and sarcThe typical presentation of ocular sarcoidosis in this age group is bilateral anterior uveitis, which can be either granulomatous or non-granulomatous Table .In case of anterior granulomatous as well as non-granulomatous uveitis, juvenile idiopathic arthritis (JIA) should be considered in the differential diagnosis. Indeed, although granulomatous uveitis in JIA really makes you wonder about the diagnosis, at least two papers mention that JIA-associated uveitis can be granulomatous , 50. FurAlso, TINU, although known mainly as anterior non-granulomatous uveitis, can present as granulomatous uveitis with subclinical choroidal involvement .In case of bilateral granulomatous panuveitis/posterior uveitis, the main differential diagnosis is ocular tuberculosis.In adolescents, as in adults, syphilis can be considered in differential diagnosis, although pure choroidal disease will not be syphilis.In Blau syndrome, a typical association of uveitis with arthritis and skin rash can guide the diagnosis. It is worth noticing that skin involvement and arthritis in EOS differ from the ones in JIA  . HoweverDiagnosis of ocular sarcoidosis recommended by IWOS, unless biopsy-proven, relies on various clinical and investigational criteria Table . Most ofChest X-ray and/or chest computed CT scan are the mainstay imaging examinations in the diagnosis of granulomatous uveitis. Their role is to detect pulmonary changes characteristic for pulmonary sarcoidosis or tuberculosis. The findings that can support sarcoidosis diagnosis are bilateral hilar lymphadenopathy (BHL) and/or parenchymal lung changes. High resolution CT or CT with contrast were also described in patients with ocular sarcoidosis , 53.In diagnosing presumed or probable ocular sarcoidosis, BHL and parenchymal lung changes are the criteria recognized by IWOS 2019. Chest X-ray and CT differ not only in their sensitivity and specificity in detecting this sarcoidosis related pulmonary features but also in availability and cost of the examination.Chest X-ray still plays a role and its biggest advantage is low cost and availability. Chest X-ray was shown to be the second, after tuberculin skin test, most contributory investigation among the first step's systematic tests in patients with uveitis. Chest CT was placed among the second step's systematic tests in patients with uveitis, and among these it was a second most often contributory investigation, after HLA-B27 .Chest CT was shown in many studies to have higher sensitivity (with similar specificity) in detecting BHL than chest X-ray. Nevertheless, for diagnosis of ocular sarcoidosis, even chest radiograph with its sensitivity 68% accompanied by specificity 96%, had high enough sensitivity to be considered as sufficient evidence of sarcoidosis among patients with uveitis . In the BHL, either by chest CT scans or chest X-ray, was found to be the most sensitive investigational finding in a study evaluating first IWOS ocular sarcoidosis criteria from 2009 .Interestingly, BHL frequency is shown to significantly differ in an age-related manner in patients with uveitis associated with sarcoidosis. In Japan, older patients (> 65 years) with OS had BHL detectable in 52% and younger patients (≤ 65 years) in 78% of cases .CT is also more sensitive in detecting parenchymal lung changes, another typical manifestation of pulmonary sarcoidosis . ParenchParenchymal lung changes seen in chest CT scans can be seen both in sarcoidosis and tuberculosis. In one study, parenchymal involvement among patients with uveitis was observed more frequently in patients with tuberculosis than with presumed sarcoidosis . AllegriThe sensitivity and specificity of various findings in chest imaging modalities in diagnosis of ocular sarcoidosis in children or in Blau syndrome is unknown.However, in children of mean age 13 years with all types of sarcoidosis, bilateral hilar lymphadenopathy was shown to be the most frequent chest X-ray manifestation (78%) .In one study, among 13 children (median age 12 years old) with sarcoidosis-associated uveitis, none of the 3 patients with definite, biopsy-proven sarcoidosis in whom chest X-ray was performed, had chest X-ray consistent with sarcoidosis . In the In Blau syndrome, pulmonary involvement with interstitial lung disease (ILD) is rare but can occur , 60. TheBoth chest X-ray and chest CT scan play an important role in detecting BHL and parenchymal lung changes that can support diagnosis of suspected ocular sarcoidosis in adults. They were proven to be the most sensitive investigations supporting diagnosis of OS in adults. In children, who have more often extrapulmonary sarcoidosis, there is no comparative data on chest X-ray and chest CT scan contribution to diagnosis of ocular sarcoidosis.18F-fluorodeoxyglucose positron emission tomography (FDG PET CT) and Gallium-67 scintigraphy are nuclear medicine imaging methods that use radiopharmaceuticals (FDG) or radioactive isotopes to detect increased inflammatory activity. 18F-fluorodeoxyglucose positron emission tomography indicates increased glucose uptake by macrophages and lymphocytes which indicates active sites of inflammation .Although FDG PET CT has gained much more attention in the research in the last decade it is still not easily accessible everywhere, where Gallium-67 scintigraphy can still have its place. FDG PET CT advantages over Gallium-67 scintigraphy are increased contrast and resolution . FDG PETNeither FDG PET CT nor Gallium-67 scintigraphy is recommended in standard systemic sarcoidosis workup. However, in cardiac sarcoidosis FDG PET CT is a second choice in a lack of cardiac MRI , 65.Both Gallium-67 scintigraphy and 18F-fluorodeoxyglucose positron emission tomography are included in revised criteria for ocular sarcoidosis IWOS.FDG PET CT’s main advantage is that it is a single whole-body examination that can detect various extrapulmonary sites of sarcoidosis . In FDG Opinions vary concerning the utility of FDG PET CT in ocular sarcoidosis in adults. Chauvelot et al., showed that FDG PET CT enabled the diagnosis of intraocular sarcoidosis even in patients with a normal CT scan . HoweverFDG PET CT’s sensitivity and specificity for ocular sarcoidosis are respectively 85.7% and 95.5%. Positive and negative predictive values for FDG PET CT for ocular sarcoidosis were also calculated to be 85.7% and 95.5% .As far as gallium-67 scintigraphy is concerned, there is considerably less data related to ocular sarcoidosis. The combination of elevated ACE and a positive 67GA scan increased the diagnostic specificity to 100% without affecting sensitivity (73%) in patients with suspected ocular sarcoidosis and normal chest radiographs .FDG PET CT in pediatrics is a valuable diagnostic tool for fever of unknown origin (FUO)  leading In children, a variety of strategies are possible to reduce the radiation dose while ensuring image quality . These iUsing ultra-low dose protocols in total body FDG PET CT is possible in children and should have special attention. Recently an ultra-low dose infection imaging using FDG PET CT was reported to be performed without sedation even in a newborn .In diagnosing bilateral uveitis of undetermined origin in children, FDG PET/ ultra-low dose CT provided important information for final diagnosis in 30% patients . In thisNo studies were found on FDG PET CT in patients with Blau syndrome.FDG PET CT and Gallium-67 scintigraphy have their place in diagnosing OS and abnormal intake and these imaging modalities are among the revised 2019 IWOS criteria in adults. In children, who have more often extra thoracic sarcoidosis presentations, a single whole body FDG PET/ ultra-low CT could be useful for final diagnosis in indeterminate uveitis as it can indicate places accessible for biopsy that are out of scope of thoracic CT. Nowadays, many strategies can be used to reduce the radiation dose, including PET/CT with ultra-low dose CT protocol which should be of special consideration in children.Systemic sarcoidosis diagnosis is supported by a CD4/CD8 ratio > 3.5 and lymphocytosis > 15% in bronchoalveolar lavage (BAL) fluid. However, BAL lymphocytosis alone is not specific for sarcoidosis as it is present in many other disorders, including hypersensitivity pneumonitis, nonspecific interstitial pneumonitis, or organizing pneumonia , 78. UsiOther authors cited by Kraaijvanger et al. showed a wider range of BAL CD4/CD8 ratio sensitivity (54—80%) with lower specificity (59—80%) .In revised IWOS criteria, elevated CD4/CD8 ratio (> 3.5) in bronchoalveolar lavage fluid (BAL) was recognised as one of systemic investigations for diagnosing suspected ocular sarcoidosis. The sensitivity and specificity of CD4/CD8 ratio (> 3.5) in BAL in diagnosing ocular sarcoidosis was not reported in the literature. However, there are studies showing its importance in diagnosing ocular sarcoidosis even in patients with normal chest imaging  includinIn children as well as in adults, BAL can be performed under sedation and topical anesthesia and using flexible bronchoscopy . HoweverThere is no data on the utility of CD4/CD8 ratio (> 3.5) in BAL in ocular sarcoidosis in children or in Blau Syndrome.In adults, testing CD4/CD8 ratio (> 3.5) in bronchoalveolar lavage fluid (BAL) can be considered in diagnosing suspected ocular sarcoidosis, as stated in revised IWOS criteria. However, its character needing at least sedation and topic anesthesia do not place it as a first-choice diagnostic examination. Furthermore, in children, it can be even less recommended due to lack of data regarding its utility in ocular sarcoidosis.ACE, studied in sarcoidosis since 1975, is the best-known serum biomarker in this disease. Serum ACE is an acid glycoprotein converting angiotensin I into angiotensin II. In the context of sarcoidosis ACE is produced by activated alveolar macrophages and correlates with granulomas burden . ElevateElevated sACE is one of the eight IWOS systemic investigations recommended as criteria for probable ocular sarcoidosis. However, it is not needed for definite or presumed ocular sarcoidosis (IWOS criteria 2019) nor for SUN Criteria for Sarcoidosis-Associated Uveitis) , 38. sACElevated levels of sACE can be found not only in sarcoidosis but also in several other diseases, among which some can also have ocular manifestations eg. tuberculosis, leprosy, diabetes mellitus and histoplasmosis. sACE levels can be influenced by ACE inhibitors, corticosteroids use and cigarette smoking , 62, 83.Notably, it was recently shown that genotype influences the sACE levels and some researchers advise to take ACE gene polymorphism into account while interpreting normal sACE levels for individuals. Taking into account the insertion (I)/deletion (D) polymorphism in the ACE gene can influence interpretation of 8,5% of measurements by either elevating or normalizing ACE values in patients with confirmed or suspected systemic sarcoidosis .Sensitivity and specificity of sACE in ocular sarcoidosis varies among the studies. However, all recent studies are compatible with the fact that sACE has lower sensitivity than specificity, with sensitivity 48% and specificity 96% , 85, 86.To increase the sensitivity of sACE as a biomarker in ocular sarcoidosis, the combination with other biomarkers was tested leading to better sensitivity while keeping high specificity. The combinations of sACE and chest radiography, lymphopenia or sIL2R were studied and showed the following changes in sensitivity of combined examinations vs sensitivity of sACE alone. Combination of sACE and chest radiography showed increase of sensitivity (70% vs 30%)  as well The standard cut off value of sACE for adults is 68 U/L. However, the optimal cutoff point for sACE levels in the population with uveitis was calculated to be 51 U/L . The norIn a recent study of ocular sarcoidosis patients, the mean serum levels of ACE were 49.17 ± 29 IU/L versus 27.4 ± 15.34 IU/L in the control group of non-granulomatous  uveitis patients .n = 27) we know that ACE was elevated in 74% of sarcoidosis pediatric patients and among all patients in the study 77% children had uveitis [Although, there is no study that determines sensitivity or specificity of sACE in children with ocular sarcoidosis, this biomarker is used in pediatric clinical practice. From a study in Louisiana on childhood sarcoidosis  pediatric patients diagnosed with sarcoidosis-associated uveitis had elevated ACE levels [In one study, all  in the control group (n = 30) [Normal lysozyme values are 9.6–17.1 mg/L for all ages and mean serum lysozyme levels was 39.92 ± 55.5 mg/L in the ocular sarcoidosis group versus 10.5 ± 5.8 mg/L ((n = 30) .There are no studies stating lysozyme sensitivity and specificity in ocular sarcoidosis in children. In one study, among 13 children with probable, presumed, or definite sarcoidosis 5 patients had elevated lysozyme levels .Serum lysozyme normal values in children are the same as for adults (9.6–17.1 mg/L) , althougSerum lysozyme is among systemic investigations criteria for diagnosing probable OS according to revised IWOS. It has high sensitivity in diagnosing OS and some studies find it even more useful than sACE. It was shown to be elevated in some pediatric patients with OS.Lymphopenia or lymphocytopenia is the blood lymphocyte count below an age-appropriate reference.Lymphopenia can occur in many conditions and among them are: steroid therapy, autoimmune disorders like lupus erythematosus, infectious diseases like tuberculosis, AIDS and viral hepatitis. Significant lymphopenia (below 1000 cells/μL) was shown to be an independent predictor of sarcoidosis in new patients presenting with uveitis .In IWOS 2019 criteria lymphopenia (< 1000 cells/μL) was added among the diagnostic criteria of ocular sarcoidosis, as the peripheral blood lymphocyte count is a simple, non-invasive test that is readily performed in patients with uveitis (IWOS 2019).Lymphopenia was observed in 35.1% patients with ocular sarcoidosis in a German study  as well 9/L) and in children younger than 2 years 3000 to 9500/mcL (3 to 9.5 × 109/L) [9/L). The range of normal lymphocyte values in teenagers (12-18y) approach adults’ norms  [9/L) and relative lymphopenia < 1500/mcL  [Although, different laboratories may have slightly different normal values, in many publications the normal lymphocyte count in adults is 1000 to 4800/mcL (1 to 4.8 × 10× 109/L) . For chi× 109/L)  but shou× 109/L) .9/L), the lymphopenia sensitivity in diagnosing sarcoid uveitis was low (15.3%) with high specificity (96.7%) [9/L) gave higher sensitivity (75%) with lower specificity (77%) [9/L with the highest Youden index, a marker of the performance of a diagnostic test, for diagnosing sarcoidosis-associated uveitis.Sensitivity of lymphopenia as a biomarker in diagnosing uveitis can vary and can depend on the choice of lymphocyte cut-off value. There are two recent studies that calculated sensitivity and specificity of lymphopenia in diagnosing sarcoid uveitis but the results differ. When considering the cut-off value of severe lymphopenia  . Howeverty (77%) . In the There is no study showing the frequency of lymphopenia in children with ocular sarcoidosis or with patients with Blau syndrome.Lymphopenia is of importance in diagnosing suspected ocular sarcoidosis and can be detected by a simple and routinely done blood test. It is one of the criteria in revised IWOS for probable OS in adults. There is no data concerning the role of lymphopenia in diagnosing OS in children or in Blau syndrome.sIL2R is a circulating form of membrane receptor for IL2 which is shed from the surface of activated Th1 cells. Activated Th1 cells are involved in formation and perpetuation of granuloma  includinsIL2R has been studied as a potential biomarker in systemic sarcoidosis. However, its role is not yet well established and is not mentioned in the diagnostic guidelines for lung sarcoidosis . sIL2R wsIL2R was found to be elevated in 69,2% - 76.2%  patientsRecently, several studies evaluating sensitivity and/or specificity of sIL2R in ocular sarcoidosis diagnosis have been published. Two of them showed high specificity of sIL2R in ocular sarcoidosis in the Japanese population , 87. sILMeasurements of Youden index were performed in some recent studies in ocular sarcoidosis. Youden index of sIL2R (0.70) in Japanese population was higher than for other biomarkers in ocular sarcoidosis eg. ACE (0.35), KL-6 (0.26), and calcemia (0.07). The authors suggested that it can indicate superior utility of sIL2R among serum biomarkers in diagnosing ocular sarcoidosis . AlthougThe optimal sIL2R cutoff value in detecting ocular sarcoidosis found by Dutch researchers was 4000 pg/mL  and a JaThere are no studies on sIL2 in sarcoidosis nor in ocular sarcoidosis in the pediatric population.In conclusion, serum sIL2R is a promising biomarker in ocular sarcoidosis with many recent studies carried out in adult patients. Several independent groups showed that the sensitivity of serum sIL2R in diagnosing ocular sarcoidosis is higher than the sensitivity of sACE. However, there are no related studies in the pediatric population.Krebs von den Lungen-6 (KL-6), which is a mucin-like glycoprotein produced by pneumocytes or bronchiolar epithelial cells, was proposed as a marker of pulmonary cells injury or inflammation. Its elevated serum levels were found in sarcoidosis but also in idiopathic pulmonary fibrosis and other interstitial lung diseases . KL-6 waKL-6 was not tested in children with sarcoidosis nor with ocular sarcoidosis. There are several studies testing KL-6 in pediatric pulmonary diseases. One study showed KL-6 as a useful biomarker for pediatric patients with connective tissue disease accompanied by interstitial lung disease .Hypercalcemia occurs in up to 4% of the population in many health conditions, including sarcoidosis, tuberculosis and lymphomas. In systemic sarcoidosis it is present in 7–18% patients , 101, alFor diagnosing ocular sarcoidosis in adults in Japan, elevated calcium (Ca) levels had rather low sensitivity (11.8%) with good specificity (95.1%).There are no studies concerning hypercalcemia in children with ocular sarcoidosis. One case report found hypercalcemia useful in diagnosing uncommon onset sarcoidosis in a 14-year-old child without ocular involvement .Polyclonal antibody activity testing bases on an observation that there is a compensatory increase of immunoglobulins as a result of decrease of T cell activity in sarcoidosis. The serologies of four herpesviruses  were used to calculate the polyclonal activation ratio .One study showed that polyclonal antibody testing has high sensitivity (70%) and specificity (90.4%) in ocular sarcoidosis .There is no data concerning children with ocular sarcoidosis and polyclonal antibody activity.Several chemokines produced by monocyte-macrophage cell lineage were shown to be elevated in sarcoidosis and to play many different roles including T-cell attraction and promotion of Th1/Th17 differentiation .In diagnosed or suspected ocular sarcoidosis, serum levels of both CXCL9 and CXCL10 were markedly elevated and correlated with ocular disease activity and ACE level . ChemokiThere are no further studies concerning sensitivity or specificity of chemokines in ocular sarcoidosis neither in children nor in adults.Liver enzymes are not included in the revised IWOS criteria for diagnosing ocular sarcoidosis, in contrast to previous criteria from 2009. In sarcoidosis liver involvement was shown to be present in 2.5–11.5% patients  or even B-cell activating factor (BAFF) is a cytokine of the TNF family that plays a vital role in the growth and function of B cells . ElevateOne study has shown that in sarcoidosis patients elevated serum BAFF show no significant difference between patients with anterior uveitis and those without ocular involvement .There are no further studies concerning sensitivity or specificity of serum BAFF in ocular sarcoidosis neither in children nor in adults.MiRNAs are small noncoding RNAs that regulate gene expression at the post-transcriptional level and are among the circulating cell-free nucleic acids released into the serum/plasma by various tissues and cells . MiRNAs Very recently miRNA microarrays (GeneChip®) were used to investigate serum miRNA profiles in patients with ocular sarcoidosis and patients with intraocular inflammation that appears typical for patients diagnosed with ocular sarcoidosis, but in whom the results of laboratory testing do not fulfill the IWOS diagnostic criteria (suspected ocular sarcoidosis). The study demonstrated a high overlap of the differential expression of serum miRNAs in these two groups of patients .Although it is only one, small and preliminary study that does not indicate sensitivity and specificity for ocular sarcoidosis diagnosis, it can open a way to establish diagnoses in idiopathic uveitis patients. There are no studies on serum miRNA in children with ocular sarcoidosis or BS.Among other biomarkers tested in ocular sarcoidosis such as KL-6, hypercalcemia, polyclonal antibody activity, chemokines,serum BAFF, and serum microRNA. The latter.seems most promising, although further studies are needed. There is no data related to these biomarkers in pediatric patients with ocular sarcoidosis.Biomarkers tested in pulmonary sarcoidosis and not yet in ocular sarcoidosis are: chitotriosidase, serum neuron-specific enolase (NSE), Serum Amyloid A (SAA), neopterin, YKL40, sCD16 and CCL18 , 109. AmChitotriosidase is a chitinase involved in defense against chitin-containing pathogens . The enzOcular biopsies can be divided into intraocular biopsies including vitreous fluid and ocular adnexa biopsies such as conjunctival biopsy.Intraocular biopsies are really performed because of its invasive character. However, it is worth noticing that the CD4/CD8 ratio in the vitreous fluid showed high sensitivity (100%) and specificity (96.3%) for the diagnosis of ocular sarcoidosis in one study . AnotherIn very rare cases, choroidal or subretinal biopsies in 27-Gauge pars plana vitrectomies are performed . This isConjunctival biopsy is less invasive, however, not commonly used. Conjunctival biopsy may be positive in approximately 50% of patients with sarcoidosis . The dirFurthermore, several studies mention conditions for increased positive conjunctival biopsy yield in diagnosis of sarcoidosis. Spaide et al. reported that the conjunctival biopsy was more likely to be positive in patients with conjunctival follicles, ocular abnormalities consistent with sarcoidosis, and in patients with pulmonary infiltrates on chest X-ray . To incrConjunctival biopsy is very interesting from a cost-effectiveness point of view. While positive results of conjunctival biopsy were like mediastinoscopy, the cost of conjunctival biopsy was ten times lower.The data on conjunctival biopsies in children in sarcoidosis is limited to case reports. One case report presented a 10-year-old female patient with conjunctival deposits but with no other ocular or systemic complaints, in whom conjunctival biopsies proved systemic sarcoidosis . AnotherIn Blau syndrome, conjunctival biopsies proved conjunctival granulomas in 2 adults and one 10-year-old child .Although highly sensitive and specific for sarcoidosis, the CD4/CD8 ratio in the vitreous fluid, can rarely be performed in everyday clinics. Conjunctival biopsy is a minimal invasive and cost-effective examen that should not be forgotten especially in patients with suspected ocular sarcoidosis and conjunctival follicles.In case of the typical triad of Blau Syndrome  genetic testing can be performed not only in children below 5 years of age but also in older patients. Although the median age at onset of eye involvement in Blau Syndrome is 5 years   in studiTo diagnose Blau syndrome or EOS, confirmation of mutation in NOD2 gene is needed and genetic counseling for patients is recommended. Traditionally, Blau syndrome is recognised if there is a family history of the disease and EOS if the mutation is sporadic . HoweverMutations that are most frequently present in Blau Syndrome are R334W and R334Q , 26 and Diagnosing ocular sarcoidosis with onset in adulthood does not need genetic testing and is based on international criteria Tables  and 2 77, 38.The diagnostic criteria for ocular sarcoidosis in children are not yet clearly stated.Of interest, single nucleotide polymorphisms (SNPs) in HLA and non-HLA genes are known to be associated with ocular sarcoidosis. HLA-DRB1*04:01 is known to be associated with OS in European-Americans . VariantIn this article, we review the clinical manifestations, differential diagnosis and diagnostic modalities of sarcoidosis-associated uveitis/ocular sarcoidosis in adults and children. We also refer to Blau Syndrome and Early Onset Sarcoidosis, in which uveitis typically starts in children under 5 years of age.Clinical features of sarcoidosis-associated uveitis (SUN criteria) or ocular sarcoidosis (revised IWOS criteria) are well studied in adults but data concerning children older than 5 years is sparse.In adults and children over 5 years of age ocular sarcoidosis is typically bilateral with panuveitis or anterior uveitis as a predominant site of inflammation depending on the study. Panuveitis is more frequent in Asian populations and most common in BS/EOS. In adults, granulomatous uveitis was observed in about half of the patients, often with mutton-fat keratic precipitates. In children over 5 years old it is possible that non-granulomatous uveitis is more frequent. In all age groups, multifocal choroiditis is the most frequent posterior presentation, followed by periphlebitis. In BS/EOS posterior synechiae and peripapillary nodules are more common than in other forms of sarcoidosis.Differential diagnosis should exclude especially ocular tuberculosis and syphilis. In children ocular sarcoidosis should be differentiated with JIA-associated uveitis and TINU.There are many diagnostic modalities supporting the diagnosis of ocular sarcoidosis but none of them is ideal or self-sufficient. Definite ocular sarcoidosis diagnosis can be stated only in case of biopsy proven lesions with compatible ocular involvement. For presumed and probable ocular sarcoidosis diagnosis performing systemic imaging or checking for serum biomarkers is needed.If biopsy is not possible, bilateral hilar lymphadenopathy (BHL) is the next most important test. CT has higher sensitivity than chest X-ray in detecting BHL in adults. Serum ACE and lysozyme are well documented as ocular sarcoidosis biomarkers. Availability of lymphopenia is its major advantage although its sensitivity is not high. There are several very promising studies on sIL2R as an ocular sarcoidosis biomarker showing its higher sensitivity even than ACE. In some patients, conjunctival biopsies or vitreous biopsies can be considered.There is very little data concerning the utility of diagnostic tests in ocular sarcoidosis in children, therefore a detailed comparison with adults is not possible. In contrast to adults, in children, extrapulmonary sarcoidosis is more frequent. Therefore, searching for biopsy accessible peripheral lymph nodes by the means of FDG PET/CT (with ultra-low CT protocol) could be an interesting option especially in children with bilateral uveitis with negative chest imaging. Blau Syndrome and EOS are, respectively, the familial and sporadic forms of the same monogenic autoinflammatory disease that needs genetic testing to be confirmed. A search for NOD2 gene mutation should be considered if a typical triad of arthritis, rash, uveitis is present.


The graphene works as channels while MoS2 functions as passivation layers. The weak hysteresis of the device suggests that the MoS2 layer can effectively passivate the graphene channel. The characteristics of devices with and without removal of MoS2 between electrodes and graphene are also compared. The device with direct electrode/graphene contact shows a reduced contact resistance, increased drain current, and enhanced field-effect mobility. The higher field-effect mobility than that obtained through Hall measurement indicates that more carriers are present in the channel, rendering it more conductive.We demonstrate in-plane gate transistors based on the molybdenum disulfide (MoS Despite its zero-bandgap nature, the high mobility of the first discovered 2D material, graphene, have demonstrated its potential in the application of radio-frequency devices11. On the hand, since the unique material characteristics of 2D materials can be observed in a few atomic layers, devices with ultra-thin bodies can be fabricated on these materials, making them a promising candidate for advanced electronics with reduced linewidth15. However, the characteristics of thin body also hinder the practical usage of 2D materials. With an active region down to nanoscale, little room is left for 2D channels vertically. Since the top gate scheme is the most common device architecture adopted for field-effect transistors (FETs) in industry, a poor dielectric/2D-material interface can easily degrade the performance of thin 2D transistors18. One possible approach to solve this problem is to use dielectric 2D materials such as hexagonal boron nitride (h-BN) as the interface layers to the SiO2 dielectrics. Due to the atomically flat surface and wide bandgap value of h-BN, superior device performances are obtained for such bottom-gate graphene transistors20. The other possible solution may be the adoption of different device architecture without dielectric/2D-material interfaces. In previous works, it has been shown that transistors with in-plane gates can be realized on compound semiconductors22. This device architecture may lower the influence of dielectric/2D-material interfaces on 2D channels.The high mobility and bright luminescence of two-dimensional (2D) materials are their major advantages in device applications2, h-BN is also adopted as the passivation layers to 2D material devices24. However, due to the high growth temperatures of h-BN, sequential mechanical exfoliations of different 2D materials instead of epitaxial growth are usually adopted for such hetero-structures, which may introduce additional contaminations to the 2D material interfaces. Nevertheless, the results still demonstrated that a less conductive 2D material may act as the passivation layer for the 2D channel. Together with in-plane gates, the 2D channel passivated by epitaxially grown 2D materials may exhibit optimized performances. In this work, we fabricated and characterized in-plane gate transistors (IPGTs) based on molybdenum disulfide (MoS2)/graphene hetero-structure27. IPGTs with a 300 nm gate-channel separation were fabricated on the MoS2/graphene film using the e-beam lithography. To further improve the device performances, we utilized atomic layer etching (ALE) technique to remove the MoS2 beneath the source/drain electrodes. In this way, the electrode metal would contact the graphene channel directly. The MoS2 passivation can effectively isolate the graphene channel from the environment. A field-effect mobility of the device similar to that from Hall measurements indicates that the device performance is well maintained using in-plane gates.However, without top covering, the channel is exposed to the atmosphere. Water or oxygen molecules in air may attach to the 2D channel and degrade the device performance. A passivation layer is still necessary to protect the channel from environments. Without extra chemical bonding, passivation based on another 2D material may bring the least impact to the 2D channel. Similar to being interfacial layers on the SiO25. Nevertheless, compared with the counterparts grown on copper foils, the graphene films grown on sapphire substrates require no transferring processes and can further simplify the device fabrication. Therefore, this growth scheme is suitable for the architecture of IPGTs. The fabrication steps of the in-plane gate graphene transistor are illustrated in Fig. 2 V−1 s−1, respectively. The field-effect hole mobility is slightly lower than the value commonly observed for the directly grown graphene films via the Hall measurement , which may be attributed to the water or oxygen attachment from the environment. The results suggest that although the in-plane device architecture may avoid the influence of dielectric layers on the graphene channel, the influence from the atmospheric condition is un-avoided with the graphene channel exposed to air. On the other hand, when the device is operated under reverse bias condition, significant Dirac point shift from 30 to − 4.0 V is also observed in Fig. oC28. The results demonstrated that the water molecules in the atmospheric condition attach to the graphene surface and influence the device performances. In this case, when the graphene transistor is under operation, the dipoles of absorbed water molecules are oriented along the direction of applied gate bias, which increases the local electric field near dipoles and therefore, increase the carrier density in the graphene channel29. In this case, a clear hysteresis loop is observed for the in-plane gate graphene transistor without a passivation layer.For demonstrating the feasibility of 2D materials on the architecture of in-plane gate transistors (IPGTs), a graphene film grown directly on sapphire substrates is prepared. The Raman spectrum of the sample is shown in Fig. mentclass2pt{minimmentclass2pt{minim28. Since dielectric materials such as Al2O3 may bring additional influence to the graphene channel and do not conform to the concept behind IPGTs, the most promising candidate for passivation layers should be other less conductive 2D material layers. In previous publication, we have demonstrated that by sulfurizing pre-deposited Mo films, MoS2 layers could be grown on graphene surfaces30. However, since the Ar plasma in the radio-frequency sputtering system may bring additional damage to graphene films, we formed the MoS2/graphene hetero-structure by sulfurizing the MoO3 film deposited with the thermal evaporator. The preparation procedure of the MoS2/graphene hetero-structure is disclosed in the “2/graphene hetero-structure with different measurement ranges corresponding to the characteristic Raman peaks of graphene and MoS2, respectively, are shown in Fig. 2 Raman characteristic peaks indicates that the MoS2/graphene hetero-structure is formed after the sequential growth of graphene and MoS2. The difference ∆k between the two Raman peaks of MoS2 is 21.3 cm−1, indicating the presence of bi-layer MoS2 after the sulfurization procedure31. On the other hand, the similar D/G peak ratios before and after the sulfurization procedure of the deposited MoO3 film (around 0.4) suggest that the possible damages introduced during either the MoO3 deposition or the sulfurization procedures are almost negligible. Although the electrical properties of MoS2 will be degraded in air32 in the IPGT architecture, it is not a concern here since the MoS2 layers are the passivation layers instead of channels. Therefore, the degradation of the MoS2 layers in the ambient condition may not influence the performance of the device. Following the similar device fabrication of standalone graphene IPGTs  of multi-layer MoSmentclass2pt{minimIn addition to the current enhancement due to the more significant accumulation of the carrier in the channel, the field-effect mobilities become 2–3 times higher than the Hall-effect ones from graphene grown on the sapphire. We note that the mobility extracted from the Hall measurement reflects the transport properties without significantly altering the intrinsic carrier density of graphene on sapphire. As a result, without being screened at low-density level, the scatterings due to lattice imperfections such as charged impurities or structural defects can all impede carrier drifting. In contrast, after the issue of contact resistance is resolved, the in-plane gates may easily attract more carriers into the graphene channel. The Coulomb effect from these additionally provided carriers can screen the interactions between lattice imperfections and drifted carriers. The scattering in the channel is screened more in the presence of the higher carrier density. Therefore, by increasing the carrier density through gating, not only the drain current but also the mobility is enhanced.2 layer are attracted by the positive drain voltage to the graphene channel and change the Fermi level35. The results revealed that with more complicated 2D material structures such as MoS2/graphene hetero-structures, different device applications such as photo-transistors can be demonstrated. With Eq.  was deposited on the graphene/sapphire substrate using the thermal evaporation. The sample was then sulfurized in a furnace at 850 °C to convert MoO3 into MoS230. After the definition of large-area contact electrodes through typical photolithography, IPGTs were fabricated with the aid of e-beam lithography. The electrodes composed of 50 nm Au/10 nm Ti were deposited with the e-beam evaporator. For the removal of MoS2 beneath the electrodes, we used a customer-designed low-pressure RF oxygen plasma system to perform ALE. The plasma power was kept at 20 W, and the background pressure was maintained at 0.4 Torr with a 30 sccm oxygen gas flow during the removal process. The etching time was 10 s. After this, the sample was dipped into deionized water for 10 s to detach the topmost oxidized MoS2 layer34. The current–voltage curves were taken with probes equipped with a Keithley 2636B system. Raman spectra were collected using a HORIBA Jobin Yvon HR800UV Raman spectroscopy system equipped with a 488 nm laser. The room-temperature Hall mobility was obtained with the Ecopia HMS-5000 Hall effect measurement system.The graphene films were grown by using the chemical vapor deposition (CVD) in a hot furnace at 1100 °C with the ethane precursor and Ar/HSupplementary Information.


The henipaviruses, Nipah virus (NiV), and Hendra virus (HeV) can cause fatal diseases in humans and animals, whereas Cedar virus is a nonpathogenic henipavirus. Here, using a recombinant Cedar virus (rCedV) reverse genetics platform, the fusion (F) and attachment (G) glycoprotein genes of rCedV were replaced with those of NiV-Bangladesh (NiV-B) or HeV, generating replication-competent chimeric viruses (rCedV-NiV-B and rCedV-HeV), both with and without green fluorescent protein (GFP) or luciferase protein genes. The rCedV chimeras induced a Type I interferon response and utilized only ephrin-B2 and ephrin-B3 as entry receptors compared to rCedV. The neutralizing potencies of well-characterized cross-reactive NiV/HeV F and G specific monoclonal antibodies against rCedV-NiV-B-GFP and rCedV-HeV-GFP highly correlated with measurements obtained using authentic NiV-B and HeV when tested in parallel by plaque reduction neutralization tests (PRNT). A rapid, high-throughput, and quantitative fluorescence reduction neutralization test (FRNT) using the GFP-encoding chimeras was established, and monoclonal antibody neutralization data derived by FRNT highly correlated with data derived by PRNT. The FRNT assay could also measure serum neutralization titers from henipavirus G glycoprotein immunized animals. These rCedV chimeras are an authentic henipavirus-based surrogate neutralization assay that is rapid, cost-effective, and can be utilized outside high containment. Henipavirus within the family Paramyxoviridae . Fi. Fi47]. tet) . The sG immune rabbit sera neutralized the infectivity of both rCedV-NiV-B-GFP and rCedV-HeV-GFP, as shown by the dose-response neutralization profiles in 50 titers for each of the rabbit sera against rCedV-NiV-B-GFP and rCedV-HeV-GFP are summarized in tet sera had similar and very high neutralizing titers of 1:65,820 and 1:59,457, respectively, against rCedV-HeV-GFP. Here, the HeV-sG-specific rabbit sera had higher cross-neutralizing heterologous titers against rCedV-NiV-B-GFP  with IC50 values again greater against the homologous immunized subjects (HeV), consistent with prior authentic NiV and HeV neutralization data derived from NiV-sG versus HeV-sG immunized cats [We also analyzed sera from rabbits immunized with either HeV-sG or HeV-sG tetramer (HeV-sGzed cats .There has been increased concern regarding respiratory pathogens such as NiV as a consequence of the COVID-19 pandemic . The mosopt) and the self-cleaving HHRbzA in the pOLTV5-rCedV antigenome plasmid, which improved the rescue efficiency of rCedVs. A similar strategy was used to rescue a number of other single-stranded, negative-sense RNA viruses, including NiV [In the present study, the rCedV reverse genetics platform ,21 was mding NiV ,80,81,82ding NiV . All rCeIFNA7, CCL5, STAT1, and STAT2 in primary hamster endothelial cells [Previous in vitro cell-based assays demonstrated that CedV and rCedV induced a robust IFN-β response ,20, and al cells . Here, tSeveral surrogate NiV neutralization assays using recombinant Vesicular Stomatitis Virus (VSV) as a replication-incompetent pseudovirus with a deletion of the VSV G glycoprotein gene have been developed as a tool to measure NiV neutralization at BSL-2 containment ,85,86. T50 concentrations of well-characterized NiV and HeV cross-reactive mAbs were observed between PRNTs using rCedV-NiV-B-GFP and NiV-B or rCedV-HeV-GFP and HeV. A strong and significant correlation between the overall neutralization values of the BSL-2 and BSL-4 PRNTs  is high-throughput and allows for more samples to be assayed with more replicates in a 96-well plate format, (ii) requires smaller sample volumes, (iii) is less time consuming taking less than 36 h from infection to assay completion, and (iv) reduces the requirement for other reagents such as luciferase substrate. The rCedV-NiV-B-GFP and rCedV-HeV-GFP chimeric viruses were used to assess the neutralization potencies of mAbs by FRNT assay, and the neutralization values at each mAb concentration obtained by FRNT were found to be highly correlated with those values obtained by PRNT . We furtIn summary, a surrogate henipavirus-based system for NiV and HeV using the rCedV platform suitable for use at BSL-2 containment has been developed and well-characterized. These rCedV chimeras can serve as useful tools to study NiV and HeV entry, membrane fusion mechanisms, and F and G glycoprotein interactions and aid in the discovery and development of henipavirus countermeasures. More importantly, the specificity and utility of the rCedV-NiV-B-GFP and rCedV-HeV-GFP viruses as a surrogate neutralization assay for authentic NiV and HeV to evaluate the neutralization potential of mAbs and NiV/HeV specific antisera has also been demonstrated. The rCedV chimeras will reduce the cost and technical challenges of the high-containment environment, particularly when large numbers of serum samples derived from NiV or HeV vaccine development programs will require testing and quantitation.C.C.B. and M.A. are United States federal employees and co-inventors on US and foreign patents pertaining to Recombinant Cedar Virus Chimeras, whose assignees are the United States as represented by the Henry M Jackson Foundation for the Advancement of Military Medicine, Inc. .


Staphylococcus aureus (MSSA) in the United States was first observed in New York City (2004–2007); its diffusion across the country resulted in changing treatment options. Utilizing outpatient antimicrobial susceptibility data from the Veterans Health Administration from 2010 to 2019, the spatiotemporal prevalence of potential ST398 MSSA is documented.Novel ST398 methicillin susceptible  Staphylococcus aureus emerged as a novel type globally in the early 2000s. ST398 with methicillin resistance (MRSA) was commonly associated with livestock contact, while ST398 with methicillin susceptibility (MSSA) was not. Genomic analysis indicates further contrasts between livestock-associated ST398 MRSA and human-associated ST398 MSSA, such as the varying presence of genes encoding resistance to tetracycline and erythromycin. Although ST398 MSSA was widely detected in European and Asian countries, in the United States it was first detected in New York City (NYC) and the subsequent region of greatest prevalence was New York state and the Northeast. Utilizing the phenotypic characteristics commonly associated with ST398 MSSA, prior analysis of MSSA collected nationwide in US hospitals documented an increased prevalence of potential ST398 from 2003 to 2014 with a significant negative association with distance from NYC. Molecular characterization of MSSA isolates across the United States from 2004 to 2010 similarly found a low prevalence of this phenotype outside of the Northeast.Sequence type 398 (ST398) S. aureus isolates, spatiotemporal surveillance of phenotypic profiles can indicate the prevalence and diffusion patterns of novel antimicrobial resistance varieties. Utilizing a retrospective cohort of all S. aureus positive cultures in outpatient Veterans Health Administration (VHA) settings from 2010 to 2019, we explored whether previously observed rates of potential-ST398 MSSA held steady or changed over time and whether the Northeast continued to serve as the epicenter for this phenotype.This phenotypic profile has been shown to be associated, but perhaps not exclusively, with ST398 MSSA. However, in the absence of widespread genotyping of S. aureus isolates from clinical specimens collected in outpatient VHA settings from January 1, 2010 to December 31, 2019 were analyzed. Microbiology reports were extracted from the Corporate Data Warehouse, the integrated electronic medical record system data repository of the VHA health system. We included all isolates from patients with a residential address in the contiguous United States who were ≥18 yr old at the time of specimen collection. Isolates were excluded from analysis if they were obtained at institutionalized care settings  or if they were taken after either 48 h of an inpatient admission or 72 h of an inpatient discharge. MSSA was defined as susceptibility to first-generation cephalosporins, cefoxitin, nafcillin, oxacillin, or carbapenems. Only isolates that were tested for resistance against all three relevant antimicrobial classes, clindamycin, erythromycin, and tetracycline, were included in the analysis. The first record per unique patient per month was retained. Potential ST398 was defined as resistance, including intermediate resistance, to clindamycin and erythromycin with susceptibility to tetracycline. When reported, inducible clindamycin resistance (commonly known as D-test) was also considered to be resistance.All Based on patient county of residence, MSSA isolates were assigned to Census regions: Northeast, Midwest, South, or West. Prevalence of potential ST398 MSSA was determined at county and regional scales for each month and year of the study.With the county-level monthly counts of potential ST398 as the outcome variable, a Poisson regression model with robust standard errors was fitted to account for the variation in the trends of potential ST398 between 2010 and 2019 as repeated measurements within each region. Region was included in the model as a fixed effect and an interaction term between time and region was included in the model to detect regional variability in temporal trends. The total number of MSSA infections was included as an offset variable. Baseline incidence rates and temporal trends were estimated as incidence rate ratios (IRRs), using Northeast and the year 2010 as references.The study was approved by the IRB of the University of Iowa.–2019, 235,033 outpatient specimens were positive for MSSA and 161,816 (68.85%) were tested against all three relevant antimicrobial classes. Among the first 145,304 isolates per unique patient per month, 22,919 (15.77%) were classified as potential ST398. Rates of potential ST398 rose slightly from ∼15% in the early years of the study to ∼17% in the final years . The Midwest, South, and West all had significantly lower potential ST398 MSSA than did the Northeast. When the interaction between time and region was examined, only the West had significantly increased potential ST398 from 2010 to 2019 (IRR: 1.02 [1.01–1.05]) compared to the Northeast.Models accounting for region and time showed similar patterns of potential ST398 MSSA Table . No sign In this study which includes data through 2019, similar prevalence is observed in cultures from all sites obtained in VHA outpatient settings. This suggests that potential ST398 MSSA has established stable levels of circulation in the US population.Prior analysis of sterile site MSSA cultures within VHA inpatient settings indicated an increase in potential ST398 MSSA from 2003 to 2010, with prevalence plateauing from 2010 to 2014 around 15%.While prevalence over time is relatively static, regional differences persist. Potential ST398 MSSA prevalence remained highest in outpatients residing in the Northeast, consistent with earlier studies that documented early detection of ST398 MSSA in NYC. Significant increases in potential ST398 MSSA are also observed in outpatients residing in the Western region of the United States. Long-distance spread and intense short-distance circulation of ST398 MSSA have been documented using phylogeographic analysis, pointing to migration routes and human circulation as responsible for the diffusion and establishment of this novel type.Understanding these geographic differences in potential ST398 prevalence, and its associated phenotypic profile, has important implications for empiric therapy, particularly in outpatient settings. In the Northeast, over 20% of the MSSA infections observed in outpatient settings were resistant to clindamycin and erythromycin, and the rising rates of resistance may influence clinicians in that region to change their empiric prescribing patterns based on their risk tolerance. At the same time, clinicians in the West need to be aware of the novel resistance profile that is slowly increasing in prevalence among their patients. While beta-lactam agents  are generally preferred options for MSSA, most empiric therapy decisions, particularly in outpatient settings, are made without susceptibility profile information. As such, epidemiologic trends of phenotypic change can be useful information to guide clinicians’ prescribing patterns.Sample collection was limited to the end of 2019 to avoid COVID pandemic confounding factors such as decreased outpatient visits and increased antimicrobial usage. Future work will explore potential ST398 MSSA prevalence to understand if spatiotemporal patterns have changed in VHA populations following the pandemic.S. aureus types in the non-hospitalized population.Though its patient population is older with higher male proportions than the rest of the US population, the VHA represents one of the only sources for nationwide retrospective phenotypic analysis of bacterial isolates in the United States. While the phenotypic profile used in this study may incorrectly identify ST398 MSSA, it is still useful to observe spatiotemporal trends in resistance profiles. Moreover, many studies focus on MRSA or invasive infections rather than more frequently experienced outpatient illnesses. Understanding the resistance patterns and associated genotypes occurring in MSSA and the community rather than in healthcare settings is an important indicator of the successful spread and persistence of novel