Microorganisms can survive in extreme environments and oligotrophic habitats thanks to their specific adaptive capacity. Due to its severe and contrasting climate conditions, the cold mountain desert in Eastern Pamir provides a unique environment for analyzing microbial adaptation mechanisms occurring within colonization of endolithic habitats. This study aims to investigate the composition and structure of endolithic microbial communities and analyze the interactions between microorganisms and colonized lithic substrates. Endolithic biofilms were examined using scanning electron microscopy in backscattered electron mode (SEM-BSE) and next-generation sequencing (NGS) applying amplicon sequence variants (ASVs) approach. The investigation of the V3–V4 region of 16S rRNA gene revealed that endolithic communities are dominated by Actinobacteria (26%), Proteobacteria (23%), and Cyanobacteria (11.4%). Cyanobacteria were represented by Oxyphotobacteria with a predominance of subclasses of Oscillatoriophycidae, Synechococcophycideae, and Nostocophycidae as well as the rarely occurring Sericytochromatia. The positive correlation between the contribution of the orders Synechococcales and Rhizobiales to community structure suggests that some functionally closed taxa of Cyanobacteria and Proteobacteria can complement each other, for example, in nitrogen fixation in endolithic communities. The endolithic communities occurring in Eastern Pamir were identified as complex systems whose composition and structure seem to be influenced by the architecture of microhabitats and related microenvironmental conditions. Microorganisms play a crucial role in biogeochemical processes occurring in desert ecosystems . DespiteMicroorganisms can supply barren soil with nutrients such as carbon (autotrophs) and nitrogen (diazotrophs), which can be used by other organisms ,14. At hSome microbial species are more adaptable and can colonize different habitats in a poor desert ecosystem, while others can subsist only in particular microniches; these can be seen as habitat specialists. Understanding the role of particular taxonomic and functional groups of microorganisms in limited niches could help find their application in biotechnology and medicine ,15.There are numerous studies on endolithic microbial communities from the cold desert of Antarctica ,10,16,17Our preliminary study reported the diversity of endolithic microbial communities in granites, limestones, and gneiss , while tThis study was designed to assess the diversity of endolithic microbial communities inhabiting granites and quartzites from Eastern Pamir. The study is part of a wider project targeting taxonomic and functional diversity at multiple niches in the cold mountain desert of Pamir (Tajikistan) based on the V3–V4 hypervariable region of 16S rRNA gene which allows for a comparative examination of the microbial profile along multiple ecological niches in a high mountain cold desert ecosystem. The research aims of this study were to (1) investigate the composition and structure of endolithic microbial communities; (2) study the complexity of the investigated communities ; and (3) explore cyanobacterial diversity and estimate their potential roles in the functioning of the investigated communities.The Pamir Mountains lie between the world’s highest mountain ranges, namely, the Himalayas, the Tian Shan, Kunlun, Hindu Kush, Karakoram, Suleiman, and Hindu Raj . In termSamplings were performed in July 2017. The collected samples were stored in sterile plastic bags and were divided into 4 pieces for the following analysis: the first was powdered and used for DNA extraction; the second was prepared and analyzed under an optical and scanning electron microscope (SEM); the third was kept for isolation and cultivation of cyanobacteria; the fourth was frozen and stored in the freezer. Samples selected for research were collected from the sites which are characterized by rather similar physical and chemical parameters of the soil, humidity, altitude, and annual temperature.A).Granites collected from the slopes situated close to lakes Khargush (southern part of Eastern Pamir), and Rangkul (eastern part of Eastern Pamir): TAKHG and TARG.B).Granites collected from sampling sites located nearby Lake Karakul (northern part of Eastern Pamir): TAKAG1, TAKAG2, and TAKAG3.C).Quartzites collected from the same sampling area as the granites from group B which is nearby Lake Karakul: TAKAW1, TAKAW2, and TAKAW3 .Sample groups:Most of the investigated samples were collected from mountainsides with a similar altitude (between 3901 and 4244 m a.s.l.) except for sample TAKAG1, which was collected from a sampling site located at 5019 m a.s.l. The rock pieces with green, blue-green, and brown visible colonization were implanted on Petri dishes containing WC or BG11 medium ,27 solidThe powdered rock samples were treated with liquid nitrogen and the DNA was extracted using a Soil DNA Purification Kit . The DNA extraction was conducted following manufacture protocols and was repeated at least three times for each sample. The concentration and quality of the DNA were checked with Hybrid Multi-Mode Reader . The V3_V4_341f and V3_V4_785r primers  targetinThe V3–V4 sequences identified as cyanobacterial according to the SILVA  databasev/v) and later with osmium tetroxide solutions (1% w/v), dehydrated in a graded ethanol series (from 30% to 100% v/v), and embedded in LR-White resin. Later, the resulting blocks of resin-embedded rock colonized samples were finely polished, carbon-coated, and observed using a FEI INSPECT 105 SEM microscope.Small fragments of rocks with endolithic colonization were prepared for observation under the scanning electron microscope (SEM) in backscattered electron mode (SEM-BSE) according to a procedure developed by Wierzchos and Ascaso  in the lHierarchical clustering analysis and non-metric multidimensional scaling (NMDS) have been conducted using the R environment . The alpThe endolithic communities from strongly weathered granites and less porous quartzites were rich in photoautotrophic microorganisms and were characterized by blue-green and green to brown colonization. Biofilms occurring in quartzites were smooth and softly attached to the substratum, while those from granites were mostly covered by tiny mineral particles . In the Overall, the biofilms occurring in granites were characterized by brighter blue-green color, while those biofilms which developed in the cracks of the quartzites were of a darker yellowish or dark green to brown color. The presence of yellowish pigment in cyanobacterial sheaths was also observed under a light microscope during the examination of the biofilms.The results of amplicon-based analysis of Pamirian endoliths revealed that the communities were dominated by Actinobacteria (26% on average), Proteobacteria (23%), and Cyanobacteria (11.4%) including Sericytochromatia which were represented by 0.1% (47 reads) ASVs (amplicon sequence variants). Other bacterial phyla were less abundant, sharing about 39% altogether. The percentage of bacteria identified to phylum level only was 0.5% and unassigned 0.03% .Actinobacteria were the principal contributors to the endolithic community in the TARG (group A) TAKAW2 and TAKAW3 samples (group C). Proteobacteria, Actinobacteria, and Bacteroidetes were abundant in the samples from group B and quartzites from group C (TAKAW1). The distribution of Oxyphotobacteria was irregular, with the highest percentage in samples TAKHG (27.3%) and TAKAG2 (26.6%). No statistically significant correlation was found between Cyanobacteria and other bacterial phyla.The results of non-metric multidimensional scaling, based on the structure of microbial communities, revealed that endolithic communities from studied groups were sharing only some ASVs, while most of the sequence variants seem to be group-specific. The NMDS plot showed a triangle-like arrangement of the communities’ structures. Thus beta-diversity analysis did not reveal a high resemblance between particular communities. .Desmochloris halophila (70 representative sequences), and Picocystis salinarum (11 representative sequences). Some sequences which could not be further classified were identified as “chloroplast” only.The total number of representative sequences originating from chloroplast DNA was 587. Some of the sequences belonged to Chlorophyta, Alpha diversity metrics calculated for studied communities demonstrate significant differences between the communities . The ShaThe clustering based on the alpha diversity revealed the presence of two main clusters of communities . ClusterCyanobacterial communities were represented by 26 groups of amplicon sequence variants (ASVs). The distribution between the communities was mosaic . The phyr = 0.76 , however the correlation index was only calculated for eight studied communities here. The community TARG that was characterized by the absence of Synechococcales, but a greater contribution of Oscillatoriales and Nostocales according to the SILVA database contained also only 1% of Rhizobiales.According to the SILVA database, the contribution of the order Synechococcales was high compared to those mainly consisting of species that can fix atmospheric nitrogen, such as Nostocales and Chroococcidiopsidales . The samThermosynechococcaceae uncultured were located on the branch of Chroococcopsis gigantea with a like weight ratio of 0.9. The sequences of Aliterella CENA 595 according to the SILVA database were mapped on the branch of Synechocystis sp. with like weight ratio 1 (maximum). The results of the phylogenetic placement of the sequences with a low like weight ratio (about 18% with like weight ratio under 0.2) suggested the possibility of a novel taxa of endolithic cyanobacteria. Sequences belonging to one of the ASVs identified as “chloroplast” were placed on a branch of Chlorogloea microcystoides with a 0.83 weight like ratio.Analysis of cyanobacterial sequences placed on the Cydracil phylogenetic tree revealed that the sequences identified as Oscillatoria, Phormidium, and Leptolyngbya) and different phyla with a high abundance of Proteobacteria (mainly represented by Gammaproteobacteria) and Bacteroidetes. In two smaller clades (red and blue clades) representatives of Oxyphotobacteria  were associated with different genera of Actinobacteria and Alphaproteobacteria. Oxyphotobacteria (Cyanobacteria) from different orders were dispersed between the three biggest clades. The smallest clade (marked yellow in Aliterella genus occurred in the clade with the genus Rubrobacter (including Rubrobacter radiotolerans) and Frankiales bacterium (Actinobacteria). Generally, communities were diverse and represented by different types of ASVs belonging to the same classes or phyla; therefore, a clear pattern of microbial associations has not been detected.The analysis of microbial association at the endolithic niche revealed the presence of four main clusters of endolithic communities . The bigThe results of 16S metabarcoding were compared with morphological analysis of isolated and cultured cyanobacteria in sampled communities.Chroococcidiopsis-like taxa prevailed in cyanobacterial microbial communities in both types of mineral substratum, the granites, and quartzites. The granites were inhabited by more diverse cyanobacterial morphotypes than quartzites . Other less abundant morphotypes were Microcoleus-like (TAKHG and TAKAW1), Synechococcus-like (TAKHG and TAKAG2), Gloeocapsa- and Phormidium-like cyanobacteria (TARG), and Cyanosarcina-like cyanobacterium (TAKAHG)  ix.The study using the SEM-BSE technique revealed the presence of two types of microbial communities based on dominants: (1) “cyanobacteria-dominated” communities , TARG.Aliterella which belongs to Chroococcidiopsidaceae and was present in the samples TAKAW2 and TAKAW3.The SEM-BSE analysis allowed the identification of different lithobionthic niches; three types of endolithic colonization are differentiated: chasmoendolithic , TARG. ILichen-dominated communities colonized granite and quartzite substrates in two samples  , althougThe communities from group B granites were characterized by a similar contribution of Actinobacteria, Proteobacteria, and Bacteroidetes. However, these phyla were represented by the different families in individual samples. The Eastern mountains represents an environment rich in diverse rocks with different architecture and unique chemical properties that are colonized by cyanobacteria. We detected some similarities in the composition of Eastern Pamir communities to the endolithic communities from the cold desert of Antarctica ,14,42, oThe granites, characterized by heterogeneous porosity, were colonized by more diverse cyanobacterial communities than quartzites, with similar physicochemical properties. Bioreceptivity, weathering processes, and the microhabitat architecture of rocks are significant factors in shaping the structure of endolithic communities ,46. The However, some similarities between the colonization of granites and quartzites were also found. Lichens and coccoid cyanobacteria were detected in both lithic substrates and the dominance of certain taxonomic groups were not associated with a specific substrate. The 16S-based structure of endolithic cyanobacterial communities demonstrated that the quartzites were dominated by specific ASVs, while the contribution of representative cyanobacterial taxa occurring in granite’s inhabiting cyanobacteria was more balanced. The dominance of aggregates of coccoid cyanobacteria in endolithic communities has been previously reported for hot deserts such as Atacama ,44,45 orContrasting differences and similarities of the composition and structure of the communities could be explained by the fluctuating chemical composition within the same type of rocks ,9. HowevSlight differences detected in the color of studied biofilms developing in both types of mineral substratum could be considered as an adaptation to the colonization of rocks with different light regimes reaching the different microhabitats. The dark yellowish color of the biofilm TAKAW3 was detected by light microscopy, suggesting the presence of some accessory pigments in cyanobacterial sheaths. One of the UV-protecting metabolites in cyanobacterial sheaths is scytonemin. The production of this pigment was considered a strategy gained by lithobionths to cope with UV-radiation, especially for those occurring in more transparent types of rocks such as quartzites . SimilarDifferent biotic interactions are visualized by SEM-BSE concerning the microbial aggregates observed in endolithic habitats from Eastern Pamir. Microorganisms develop naturally in consortia in the environment, sharing some functions which are essential to survive in deserts or needed to cope with harsh conditions . The invInsight into the diversity and distribution of microbial taxa in extreme and in nutrient-limited environments, as well as an exploration of the relationships between abiotic and biotic components at different niches, could be key elements of understanding the functional diversity and the role of organisms in ecosystems ,48. Morp


Next, the promising applications of graphene-based electrodes in promoting this two-electron oxygen reduction reaction are considered and this is followed by an account of the various graphene-based materials that have been used successfully to give highly efficient graphene-based cathodes in electro-Fenton. In particular, graphene-based composites that have been combined with other carbonaceous materials, doped with nitrogen, formed as highly porous aerogels, three-dimensional materials and porous gas diffusion electrodes, used as supports for iron oxides and functionalised with ferrocene and employed in the more effective heterogeneous electro-Fenton, are all reviewed. It is perfectly clear that graphene-based materials have the potential to degrade and mineralise dyes, pharmaceutical compounds, antibiotics, phenolic compounds and show tremendous potential in electro-Fenton and other advanced oxidation processes.In recent years, graphene-based materials have been identified as an emerging and promising new material in electro-Fenton, with the potential to form highly efficient metal-free catalysts that can be employed in the removal of contaminants from water, conserving precious water resources. In this review, the recent applications of graphene-based materials in electro-Fenton are described and discussed. Initially, homogenous and heterogenous electro-Fenton methods are briefly introduced, highlighting the importance of the generation of H These oproducts . Conventproducts ,4,5. Theing AOPs ,6,7. 2+ to Fe3+ facilitates the conversion of H2O2 to the highly oxidising OH•, with a standard reduction potential of 2.56 V vs. SCE . Consequently, these OH• radicals can be employed to mineralise a large number of organic contaminants. The sustained production of OH• requires both Fe2+ and H2O2. The Fe2+ ions can be regenerated through the reduction of Fe3+ at the cathode (Equation (2)), provided the Fe3+ ions do not form hydroxide precipitates in the solution phase, to give a near-continuous supply of Fe2+. The H2O2 is produced by the two-electron reduction of dissolved oxygen; see Equation (3).The main principle of the E-Fenton process is summarised in Equation (1), where the oxidation of Fe2 and Fe(OH)3, the system must be operated under stringent pH control as these hydroxide species are only soluble at pH values lower than about 4.0. Therefore, the pH of water samples or effluents must be acidified and brought to an acidic pH value of approximately 2.0 to 3.0. Moreover, the OH• radicals are not continuously generated and require a supply of pure oxygen. However, the classical homogeneous E-Fenton process suffers from a number of limitations. The generation of secondary sludge, as ferric and ferrous ions in the treated wastewater, gives hydroxide precipitates and these must be removed, adding cost and reducing the overall efficiency. To limit the formation of solid Fe(OH)3O4, into a suitable electrode material. In 2+ is generated from a sacrificial anode and reacts with the H2O2 in the bulk solution. An iron salt can also be added to the cell to facilitate this reaction. In contrast, the aim in heterogeneous E-Fenton ) [2O2 is achieved at pH values between 2.0 and 3.0 [+, while the lack of protons with increasing pH reduces the rate of the reaction.As high yields of H process . Consequd oxygen ,30. Despd oxygen . In addid oxygen . The rednterface  (Equatioreaction  (Equatioion (7)) . It is g and 3.0 , with mo2O2 production rate of 7.7 mg h−1 cm−2.In an attempt to overcome these complex issues, various carbon-based materials have been considered, as these are generally cost effective and facilitate the two-electron reduction of oxygen. In particular, carbon in different forms, such as amorphous carbon, glassy carbon, graphite, fullerenes and carbon nanotubes have all been evaluated . Howeversp2-hybridised carbon atoms arranged in a two-dimensional honeycomb monolayer to produce a one-atom-thick sheet. Graphene can be produced using a variety of both top-down and bottom-up approaches, including exfoliation, sonication, ball milling, chemical vapour deposition and epitaxial growth [3, KMnO4 and H2SO4, which is widely known as the modified Hummers method [Graphene is a two-dimensional material with l growth . Howeverl growth . One of l growth ,43,44. Gs method ,46. Once2O2 in E-Fenton applications. The reduction methods employed are generally thermal or chemical reduction processes [2, giving a reduction of GO and the formation of rGO. Various reducing agents, such as borohydride, hydrazine or hydrogen iodide can be employed at room temperature or with mild heating to give rGO. However, some of these chemicals have environmental concerns and they can introduce impurities to the carbon matrix. These health and environmental issues can be overcome by using ascorbic acid as the reducing agent. This has been successfully employed to reduce GO and the resulting rGO was shown to have good conductivity [This oxidation process introduces a number of negatively charged oxygen-containing groups, such as hydroxy and carboxy groups, to the GO sheets and as a result, the GO sheets have good hydrophilic properties. GO exhibits good stability as a colloidal solution and this is very useful for solution processing approaches. The GO sheets can be subsequently reduced to give rGO, which has a much higher conductivity, and it is this member of the graphene family that is most suitable for electrochemical applications, such as the reduction of dissolved oxygen to produce Hrocesses . The theuctivity . The eleuctivity ,50,51. F2O2. These approaches include various electrophoretic and electrodeposition routines to generate graphene-based cathodes, graphene-based materials deposited from slurries or suspensions at graphite or carbon felt electrodes which act as supports, graphene-based inks, porous graphene-based aerogels, three-dimensional graphene-modified electrodes, heteroatom doped graphene composites, graphene composites combined with iron oxides and graphene-based diffusion electrodes. These are now described and discussed in the following sections.Different approaches have been used in forming graphene-based cathodes in an attempt to give enhanced and selective production of HOne of the most common approaches is to use carbon or graphite felt as a support for GO or rGO as these three-dimensional felt cathodes have low cost, high surface area, excellent conductivity and high porosity. The exfoliated GO/rGO can be deposited at the carbon or graphite electrodes using electrophoretic deposition , coated 2O2 with low energy consumptions of 9.7 kWh kg−1 [−1 [Indeed, these electrochemical approaches, employing combinations of electrochemical exfoliation and electrophoretic deposition, followed by the reduction of GO to rGO, have been used to coat carbon felt electrodes , while ikWh kg−1  and 3.08kg−1 [−1 , sulfadikg−1 [−1 . 2O2 (Equations (8) and (9)). For example, Gao et al. [2O2; see Equation (12).On the other hand, graphene-containing slurries can be readily formed using exfoliated or electrochemically exfoliated GO and these have been combined with quinones (AQ), which have the ability to generate Ho et al.  used GO,2O2 when GO/rGO is employed as the cathode material. Indeed, Nambi and co-workers [2O2 and the degradation efficiency of E-Fenton to quinone functional groups on electrochemically exfoliated rGO.It is well established that a number of oxygen-containing functional groups, such as epoxides (C–O–C), hydroxy (OH), carboxylic (COOH) and carbonyl groups (C=O) are present on GO nanosheets . However-workers  attribut−1 for the ferrocene-modified rGO electrode, significantly higher than the value of 0.004 min−1 obtained for the unmodified graphite felt and also higher than 0.010 min−1, which was the rate constant observed with the reduced rGO-modified felt electrode. The authors concluded that the rGO and ferrocene participated in sequential steps, with rGO facilitating the production of H2O2, while the Fe2+ centre in ferrocene catalysed the decomposition of H2O2 to form OH• and Fe3+-centred ferricenium. The same group studied the ferrocene-functionalised rGO felt electrode as an E-Fenton catalyst using rotating disc voltammetry for the removal of ciprofloxacin and carbamazepine [• concentrations of 644 µM, 264 µM and 163 µM at pH values of 3.0, 7.0 and 9.0, respectively, facilitating the removal of contaminants over a wide pH range. Graphene oxide can be easily functionalised with ferrocene ,67, and mazepine . Using r3 and Ce and deposited the hybrid composite, rGO/Ce/WO3, on carbon felt. This composite was employed in E-Fenton to remove ciprofloxacin with complete degradation within 1 h, and a mineralisation degree of 98.55% within 8 h. It was suggested that the Ce3+ catalysed the decomposition of H2O2 to give the OH• radical species (Equation (13)), while superoxide was generated from the reaction between Ce3+ and dissolved O2 (Equation (14)) and between W5+ and O2 (Equation (15)). The Ce4+ was recycled to Ce3+ through a simple reduction step (Equation (16)), while Fe2+ ions added to the E-Fenton cell also facilitated the reduction of Ce4+ (Equation (17)).Using another approach, Mi et al.  combined2O2 production and greater efficiency in the degradation of methylene blue in heterogeneous E-Fenton compared with the GO-free PEDOT:NaPSS. This was attributed to a synergistic effect between PEDOT and GO, promoting higher electron transfer rates.Graphene/GO has also been combined with conducting polymers and used in E-Fenton. In a recent study, a simple electropolymerisation method was employed to deposit poly  (PEDOT), sodium polystyrene sulfonate (NaPSS) and GO on graphite felt electrodes . This GO2O2 generated and a three-fold increase in the rate of phenol degradation. A graphene-based paste cathode has also been employed to degrade a pharmaceutical product [In addition to electrophoretic deposition and coating from graphene-containing slurries, there has been considerable interest in graphene-based inks that can be painted or printed onto substrates ,70. This product . The pas product  used ano product , and an  product , while a product .2O2 generation to give a more efficient removal of the contaminants. Interestingly, it was shown by Wang et al. [2O2. This suggests that the graphene-modified electrodes are not only more superior than the conducing graphite or carbon substrates used in their fabrication, but they may also be more efficient in producing H2O2 than carbon nanotubes. The impressive performance of the graphene-modified carbon/graphite felt electrodes is clearly illustrated in 2O2 generated is expressed differently, sometimes as rates and in other cases as mass per volume, it is evident that a graphene-based system can be employed to give the more efficient generation of H2O2 with relatively high rate constants for the removal of a variety of contaminants.In most of these approaches, the graphene-modified electrode is compared to the conducting carbon or graphite felt or carbon cloth substrate electrodes and the addition of rGO clearly enhances the rate of Hg et al. , who empPorous graphene-based composites that can be employed as aerogels, foams and as gas diffusion electrodes are interesting and these new materials are beginning to emerge in E-Fenton. 3O4 nanoparticles can be embedded and protected within the aerogel [Aerogels are three-dimensional highly porous materials and graphene-based aerogels exhibit interesting properties of high thermal stability, surface area and electrical conductivity . They ar aerogel . A numbe aerogel ,87,88,892+, EDTA-Ni. Using surface analytical techniques and adsorption measurements, the authors confirmed that the three-dimensional structure possessed homogenous macropores with a high surface area of 280.15 m2 g−1. The cathode showed enhanced electrocatalytic activity, leading to the efficient generation of H2O2 and regeneration of Fe2+. Likewise, Nazhif Mohd Nohan et al. [For example, Wen et al.  preparedn et al.  used a o2O3 nanoparticles have also been successfully fabricated and employed in the heterogenous E-Fenton system for the degradation of Rhodamine B [2O3 nanoparticles enhanced the degradation rate and this was attributed to the generation of H2O2 within the aerogel and the good diffusion and electrosorption of Rhodamine B within the aerogel to give high concentrations of the accumulated contaminant. The H2O2 was decomposed in the presence of Fe2O3 to give oxidising OH• with little quenching, as high concentrations of the pollutant were available. Chemical oxygen demand elimination rates up to 82% were observed, while a degradation rate of 99% was reached after 30 min. Low iron leaching rates were observed even in acidic media and there was no significant loss in the catalytic activity over six cycles. In a separate study, a carbon aerogel with rGO, CNT and iron oxide nanoparticles, Fe3O4, was formed successfully using a sol–gel process and then employed in an E-Fenton cell as a cathode in the degradation of methyl blue [Graphene-based aerogels containing trapped Fedamine B . The addhyl blue . The goo2O2 electrogeneration yield, degradation and mineralisation rates [In terms of three-dimensional graphene-based materials, there is some evidence that three-dimensional graphene-modified foams may be suitable in E-Fenton. In a comparison of graphene-based monolayer, multilayer and three-dimensional graphene-based foams as cathode materials in E-Fenton for wastewater treatment and the mineralisation of phenol, it was found that the three-dimensional foam exhibited the highest Hon rates . The supon rates  have sho2O2 [+ from the aqueous phase, while the conducting catalyst provides the electron, facilitating the formation of H2O2. Recently, this approach is finding applications in E-Fenton [Gas diffusion electrodes (GDE) are being used increasingly in fuel cells , batteri2O2 ,95. TheyE-Fenton ,97,98. H2O2 through the oxygen reduction reaction, for the removal of Rhodamine B [2O2. The porous structure of the graphene-based diffusion electrode provided a reaction chamber for the efficient conversion of oxygen to H2O2. Liu et al. [2O2 production was significantly improved. The apparent rate constant of dimethyl phthalate degradation was computed as 0.0322 min−1. The graphene-based diffusion electrode showed excellent recovery and could be reused. However, the degradation rate decreased slightly with increasing use and this was attributed to the blocking of the microspores and channels by iron sludge as the E-Fenton reaction proceeded. In another study, a gas diffusion electrode, consisting of a cylindrical body with a built in air diffuser, was fabricated using carbon cloth treated with PTFE and coated with electrochemically exfoliated rGO for the removal of industrial electronic wastewater [2O2 concentrations of 495 mg L−1 were achieved to give a mineralisation rate of 80% over 80 min. Gas diffusion electrodes have also been fabricated by combining rGO with Fe3O4 [3O4 particles adjacent to the electrogenerated H2O2 facilitated the efficient generation of OH• and the removal of Bisphenol A [2O2, and they have been employed in the removal of several pollutants.For example, a graphene–graphite diffusion electrode with high conductivity and electrocatalytic activity was formed for the continuous in situ generation of Hdamine B . A removu et al.  used a gstewater . High H2th Fe3O4  and by uth Fe3O4  for the phenol A . A compa2O2. In a comparative study, tert-butyl-anthraquinone (TBAQ) was used to modify four different carbon materials, carbon aerogel, CNT, carbon black and graphene-doped carbon black to fabricate gas diffusion electrodes for the production of H2O2 [2O2 production and current efficiency were achieved with the CNT-gas diffusion electrode modified with 2% TBAQ, giving 2.15 mg h−1 cm−2 compared to 1.97 mg cm−2 h−1 of H2O2 for the corresponding graphene-based material. However, rates higher than 2.15 mg h−1 cm−2 can be seen in While many studies indicate the superiority of graphene/rGO in fabricating gas diffusion electrodes, there are examples where other carbon-based systems exhibit a somewhat higher catalytic activity for the generation of H of H2O2 . In this2O2 [2O2 [The doping of graphene/rGO, using heteroatoms such as N, B, P and Fe, has been widely studied for the development of catalysts for the oxygen reduction reaction ,107. Dep2O2 , while F2O2 . Likewis2O2 [2O2 . As N ha2O2 [2O2 ,111. How2O2 [2O2 ,113. The2O2 [2O2 ,114, whi2O2 [2O2 . It has 2O2, but on reduction with NaBH4, the same materials exhibit more selectivity for the four-electron pathway [2O2 is connected with epoxy or ether groups in the N-doped rGO, while N-doped graphene composites, with abundant quaternary nitrogen species, show the selectivity of the two-electron reduction pathway; however, with pyridinic species, the four-electron pathway is preferred [The four-electron transfer pathway is normally observed in alkaline solutions , while,  pathway . Kim et  pathway  suggestereferred . These s3C nanoparticles was used for the removal of phenol, methanol, acetone, dichloromethane and diethyl phthalate from real samples [3C nanoparticles were encapsulated and protected, preventing the aggregation or leakage of the Fe, while the high-surface-area porous framework, with abundant channels and pores, provided access for the pollutants, while facilitating the formation of H2O2. A gas diffusion cathode was formed with N-doped graphene composites and CNTs for the removal of dimethyl phthalate [2O2 at a relatively low potential of 0.2 V vs. SCE. Yang et al. [•, with a high yield of 6.2 mg h−1 cm−2 of H2O2. Su et al. [2O2, reaching a value of 8.6 mg h−1 cm−2, with a selectivity of 78% at neutral pH and a low energy consumption of 9.8 kW h kg−1. In another recent study [2O2 and the generation of OH•. This N-doped graphene-modified graphite felt electrode was used in the mineralisation of 2,4-dichlorophenoxiacetic acid with an impressive rate of 88% at a pH of 7.0 after 480 min. A number of N-doped graphene-based electrodes have been employed successfully in E-Fenton. Li and Zhang  employed samples . The auththalate . The autg et al.  employedu et al.  obtainednt study , N-doped3O4 catalyst, giving a removal rate of 71%, a mineralisation rate of 51% with good stability and low iron leaching of 0.33 mg L−1. Interestingly, it was observed that the N/S-doped graphene facilitated the four-electron oxygen reduction reaction, while it was mainly the two-electron pathway that was observed with the N-doped graphene-based aerogel. While N-doped graphene appears to be superior in the promotion of the two-electron oxygen reduction reaction, Wu et al. [Graphene-based aerogels, where the graphene was doped with N and also co-doped with N and S were compared as cathodes in a heterogeneous E-Fenton cell . The higu et al.  have shoGraphene/GO can be easily combined with a variety of other components such as metal oxides , metal n2+/Fe3+ couple to catalytically decompose H2O2 into OH•, a potent oxidant . While the Fe2+ and Fe3+ are soluble in acidic solutions, with any increase in pH, for example through the four-electron reduction at the cathode (Equation (4)), insoluble iron hydroxides begin to form, reducing the concentration of Fe2+ ions that are needed for the E-Fenton reaction. This is readily seen from the Pourbaix diagram presented in 2+ and Fe3+ are shown as a function of pH. The dashed lines show the pH dependence of the oxygen reduction and hydrogen ion reduction reactions.As detailed earlier, the homogeneous E-Fenton reaction uses the Fe3+ to Fe2+ is approximately 6000 times slower than the oxidation of Fe2+ to Fe3+, which inhibits efficient recycling between the Fe2+ and Fe3+ ions [3+ to Fe2+ in the solid-state catalyst. As shown in 2O2 or OH• concentrations formed, where it is seen that relatively low leaching rates can be achieved.Moreover, the reaction rate of Fee3+ ions ,132. In e3+ ions ,133. In 2O3, Fe3O4 and FeOOH, and ferrocene, which all contain the Fe2+/Fe3+ redox pair, have been employed [3O4 [2+. This system was employed at a neutral pH to give a total organic carbon removal efficiency of 89%. Zero-valent iron has also been encapsulated within a three-dimensional graphene-based network to give a catalyst for the adsorption and degradation of sulfadiazine [2O3 as the iron source have also been employed in heterogenous E-Fenton [−1) in acidic solutions.The oxides Feemployed ,139,140,yed [3O4 ,142, FeOyed [3O4 , zero vayed [3O4  and ferryed [3O4 . In a reyed [3O4  fabricatadiazine . Iron oxE-Fenton . In this3O4, has been used more widely as the iron-containing catalyst [3O4 nanoparticles was employed for the continuous electrogeneration of H2O2 and OH•. A degradation efficiency of 98% was observed for the removal of Bisphenol A within 90 min at neutral pH with less than 1% of iron leaching [3O4 nanoparticles on GO and rGO to enhance the removal of two dyes, methylene blue and acid red. Shen et al. [3O4 hollow hybrid microspheres, while graphene oxide–Fe3O4 was employed as a heterogeneous catalyst for the E-Fenton degradation of two antibiotics, chloramphenicol and metronidazole [3O4 particles have also been utilised with N-doped graphene-based aerogels for the degradation of acetaminophen with a low iron leaching of 0.33 mg L−1 in heterogeneous E-Fenton [3O4 particles at N-doped rGO as catalytic particle electrodes. The Fe3O4/N-rGO also served as the heterogeneous catalyst, resulting in the rapid regeneration of Fe2+ and high concentrations of OH• oxidants [The iron oxide, Fecatalyst ,144. Forleaching . Akerdi leaching  have alsn et al.  have empnidazole . Fe3O4 pE-Fenton . These iE-Fenton . In a mooxidants .2/Ce0.75Zr0.25O2 composite was fabricated and used for the effective removal of ciprofloxacin. The decomposition of H2O2 to generate OH• was facilitated by the redox pair Ce3+/Ce4+ to give complete removal of ciprofloxacin within 5 h, with a mineralisation rate of 77% in 3 h [+, which then catalysed the decomposition of the electrogenerated H2O2 to give OH• and as a result the Cu+ was converted to Cu2+. The conducting catalyst layer, serving as the cathode, facilitated the reduction of Cu2+ to generate Cu+, leading to the efficient recycling of the Cu+/Cu2+ redox couple. Metal-free E-Fenton has also been recently proposed, as graphitic and pyridinic N sites on graphene-based materials appear to function as active sites for both the electrogeneration of H2O2 and the activation of OH• radicals [There is also increasing interest in finding iron-free or, indeed, metal-free catalysts for heterogeneous E-Fenton. An iron-free rGO/MoS% in 3 h . HKUST (% in 3 h . It was radicals ,126,148.2O2 is considered as an undesirable product in many research fields, such as batteries and fuel cells, tailoring the properties of the graphene-based composite with doping or by combining it with other materials to give a two-electron oxygen reduction reaction has significant potential not only in E-Fenton, but there are a number of other technologies, such as antimicrobial, medical, bleaching, gas scrubbing and refinery applications, that would benefit from the in situ generation of H2O2, eliminating the need for its storage.Graphene-based materials have attracted considerable interest, both from a fundamental viewpoint and in terms of their potential applications, and this family is one of the most studied, surpassing all other two-dimensional materials. It is no surprise that these materials are now finding applications in E-Fenton and, when combined with other carbon-based materials, they have the potential to give true iron- and metal-free E-Fenton catalysts. This would have a significant impact in terms of cost and environmental concerns. As shown earlier, rGO can be employed effectively in several ways, supported onto carbon or graphite electrodes or cloth, combined with CNTs, iron and other metal oxide catalysts, formed as aerogels and used in gas diffusion electrodes and doped with N and other elements, with N-doping appearing to be the best option in E-Fenton. It certainly appears that GO, with its very good conductivity when reduced to give rGO, high surface area, and good stability, will be used increasingly in future research in E-Fenton, leading to new and exciting developments. While the electrocatalytic generation of H2O2, while catalysing the efficient conversion of H2O2 into OH•, reducing or eliminating the release of metal ions, such as Fe3+ or Fe2+, into the water, are required. These catalysts will also need to exhibit high stability, enabling their use over multiple E-Fenton cycles, with low energy demand. This will require the development of new Fenton catalysts and graphene composites have a clear role to play in these developments, most likely in terms of composites formed using other new and emerging materials, including other two-dimensional materials, and by doping.In order for E-Fenton to emerge and be well integrated into real wastewater treatment facilities, new and more efficient Fenton catalysts are required. While heterogenous E-Fenton addresses many of the limitations of homogeneous E-Fenton, more efficient catalysts that are capable of generating high yields of HIn terms of possible new materials that could, in the future, be combined with graphene-based materials, MXenes deserve a special mention. These are exciting two-dimensional materials ,150 thatHowever, the application of graphene-based materials, as composites, or combined with other materials, and/or with doping, in E-Fenton has a number of challenges that must be overcome before significant advances are made. While remarkable progress has been made in the synthesis of graphene flakes, GO and rGO, a cost-effective large-scale synthesis is needed before these applications become a reality. The cost effective production of graphene and its oxides is required, but control over reproducibility and quality is equally important. Wet graphite exfoliation methods, including chemical and electrochemical processes, are promising in terms of scalability and cost, but concerns still remain over the reproducibility of these approaches and the quality of the final graphene product. Nevertheless, there is evidence to show that the oxygen reduction reaction is promoted at graphene edges, rather than basal planes, making these wet graphite exfoliation methods possibly suitable for the scaling up and production of graphene-based cathodes that can be employed in E-Fenton.It is also difficult to precisely control the doping levels of graphene-based materials. At another level, there are some concerns in terms of the environmental impact of GO and its potential adverse effects on aquatic ecosystems . In partNevertheless, with further advancements in the synthesis, scale-up and processing of graphene-based composites and electrodes, it is very likely that new Fenton catalysts and, indeed, metal-free Fenton catalysts can be fabricated, leading to innovations in advanced oxidation processes for the protection of water resources.


A surface plasmon resonance (SPR) sensor based on gate-controlled periodic graphene ribbons array is reported. Different from the conventional methods by monitoring reflectivity variations with respect to incident angle or wavelength, this approach measures the change in SPR curve against the variation of graphene chemical potential  at both fixed incident angle and wavelength without the need of rotating mirror, tunable filter or spectrometer for angular or wavelength interrogation. Theoretical calculations show that the sensitivities are 36,401.1 mV/RIU, 40,676.5 mV/RIU, 40,918.2 mV/RIU, and 41,160 mV/RIU for analyte refractive index (RI) equal to 1.33, 1.34, 1.35 and 1.36; their figure of merit (1/RIU) are 21.84, 24, 23.74 and 23.69, respectively. Significantly, the enhancement in the non-uniform local field due to the subwavelength graphene ribbon resonator can facilitate the detection in redistribution of protein monolayers modeled as dielectric bricks. Optical biosensors monitor variations of the scattering characteristics of incident light including intensity, phase, wavelength, and angle due to interaction between the target analyte and bioreceptor such as nucleic acid sequences, proteins, antibody, enzymes, and etc. Optical fiber with a tapered small core for single-mode operation designed to serve as a high sensitivity RI sensor was reported2. The resonant waveguide gratings were extensively reviewed3, particularly including applications of sensors for detecting the RI change on the waveguide grating surface.The emergence of the respiratory virus COVID-19 outbreak allows us to know the unprecedentedly urgent need of a rapid and accuracy diagnostic test method. Among the known diagnostic techniques, label-free optical biosensors provide high sensitivity and robustness solutions. Most importantly, such a biosensor can be integrated into a compact device and enables the possibility of bedside (or point-of-care) testing9. Additionally, enhanced absorption of surface plasmons on a periodic array of graphene ribbons for TM-polarization  was reported13. Specifically, the obvious optical absorption due to the SPR on graphene ribbons, particularly on its fundamental resonant mode, was demonstrated10.Surface plasmons are collective oscillation of the free electron gas density on the surface of noble metals or graphene. Through the electromagnetic wave coupling between photon and plasmon, the excitation of SPPs (surface plasmon polaritons) is present with its field exponentially decaying (evanescent wave) along the direction perpendicular to the surface14. A nanodevices for detection and real-time monitoring of biomolecular events using SPR and SPR imaging were thoroughly reviewed; the signal modulation schemes including angle, wavelength, amplitude and phase were discussed, as well as their coupling mechanism15. The SPR imaging techniques incorporating wavelength scanning based on solid-state wavelength filter were reviewed from the perspective of detection speed, sensitivity and portability16. RI sensor using long-range surface plasmon mode, which exhibits symmetric field pattern, was shown to have high sensitivity compared with conventional SPR ones due to low loss17. The plasmonic nanograting was employed to tailor the dispersion of plasmonic response; more specifically, the enhancement in quality (Q) factor further improves the figure of merit (FOM) of the biosensor18. The nanoplasmonic biosensor plays an important role in optical sensors because of its high sensitivity to the change in RI. More specifically, the biomolecular recognition elements on the surface of biosensors recognize and capture analyte in a liquid sample, causing an increase in the average RI on the sensor surface; several approaches for detecting the RI change were reported23. An effective design approach for achieving the high FOM of a guided-mode resonance sensor made of a grating-waveguide was numerically studied24. Furthermore, by adding a dielectric thin film having a high real part of permittivity for dramatically improving the sensor sensitivity was reported25. In their study, the guided-wave SPR structure can enhance the evanescent field at the interface between silicon and cover. Additionally, by spin-coating the GeSe nanosheets on the surface of noble metal (Au), a SPR sensor can significantly enhance its sensitively26.Moreover, the sensed medium attached to the position where SPR occurs strongly affects the resonance effect. Such a phenomenon has been widely applied in the design of biosensors for detecting the change in RI of an analyte. To mention a few, the literature focused on SPR sensors, which were designed for detecting chemical and biological species, based on the unlocalized surface plasmons propagating along wave-guiding structures were intensively reviewed27. A transmission type SPR-based sensor made of graphene nanoribbons array in infrared (IR) wavelength range was proposed to detect the wavelength shift of the resonant transmission dip due to the RI change of the analyte medium placed on the sensor surface28. A graphene-based tunable mid-IR biosensor was demonstrated on the protein characterization including both complex RI extraction and vibration fingerprints. The gate-controlled self-biasing graphene ribbons array is dynamically tuned to selectively excite the local SPR mode at different frequencies29.Regarding the biosensors made of graphene ribbons, the researchers detected multiple SPR absorption bands on graphene ribbons by changing the graphene chemical potentialThis research reports the computational design of a biosensor for RI sensing at mid-IR wavelengths. Specifically, the SPR modes supported by graphene nanoribbons are excited by evanescent wave rather than plane wave. The excitation of SPR modes in the nanoresonators (nanoribbons) enables the local-field enhancement around the slit (the region between two adjacent ribbons) edges. The placement of analyte on the surface of nanoresonators strongly affects the resonant condition of surface plasmons, particularly on the fundamental mode. As is well known, the wavelength- and angle-interrogation are the two major approaches for detecting the RI change due to a shift in SPR-curve. Significantly, in this research, graphene chemical potential scan is employed to replace the sophisticated wavelength modulation or incident angle scan. By electronically tuning the gate voltage (dc) to change the chemical potential (Fermi energy) in graphene, one can detect the smallest resolvable RI change of an analyte through the shift in SPR curve (against the variation of gate voltage). Furthermore, the tunable gate voltage can be provided by a high-resolution digital-to-analog converter (DAC) equipped with an amplifier. Furthermore, a periodic dielectric bricks array was employed to model discrete sensing mediums such as protein monolayers. The perturbation on SPR curve due to their redistribution and variation of surface percentage coverage (concentration) were also examined. Such a mechanism can be a potential candidate for designing a cost effective biosensor with acceptable performance parameters.x-axis; they are deposited on a silica , carrier relaxation time  image detection. The detail process of graphene transfer in fabricating the crystalline silicon-graphene-gate oxide structure can be found in Fig. 9 of33. Additionally, the graphene layer between Si and 34. As was reported by the authors, this device is compatible with the conventional integrated circuits process. Regarding the issue of interaction between graphene and 35, the electronic measurement showed that the single layer graphene on 29.The single layer graphene inserted between the crystalline silicon and gate oxide has been successfully implemented in the structure of a graphene photodiode-oxide-semiconductor field effect transistor (PDOSFET) shown in Fig. 8 ofy-axis) is normally incident upon the wedge surface shown in Fig. y-axis is much greater than the operating wavelength; the electric- and magnetic-fields have no variation along that direction. Therefore, a simplified 2D electromagnetic model depicted in Fig. 36 exponentially decaying along the z-axis excites the SPR modes on the graphene ribbons. Notably, because that the period of graphene ribbons array ; therefore, it is more feasible and efficient than the commonly used methods.Figure  entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaWe redraw the reflectance against chemical potential shown in Fig. entclass1pt{minimaentclass1pt{minimaV is the voltage induced on the graphene sheet.To understand the physical insight of wave process involved in the structure, the absorption against wavelength for the same biosensor employed in the previous example is calculated subject to an arbitrary analyte RI such as x-component current density  structure has a significant sensitivity enhancement. Nevertheless, due to the non-uniform evanescent field, the developed sensor can detect the redistribution of discrete analyte as shown in the later section.Furthermore, the three wavelength-selective (resonant) absorption (in blue color) occurring at 40 by fitting a sum of exponential functions to the sampled data of Returning to Fig. To evaluate the performance of this sensor, we calculate the RI sensitivity and figure of merit. The analyte under test is modeled as a uniform dielectric layer with refractive index mentclass2pt{minimThe typical RI detection resolution is around N is the bit number of a DAC and V for Returning to Table  29  to mimic their redistribution. Figure In Fig. 41. Parameter C, in fact, can also be regarded as the protein concentration. Here, C) from 0.1 to 0.05% with a step In the second example, in each unit cell the protein monolayer is positioned at the center of slit, shown in the inset of Fig. y-axis in practical manufacture; that is, the electric- and magnetic-fields are supposed to have no variation along that direction. Due to the periodicity along the x-axis (periodic graphene ribbons array), the electromagnetic fields in each uniform dielectric layer and semi-infinite medium are represented by Rayleigh expansions (or Floquet–Fourier series)45. Owing to the zero thickness approximation of the graphene grating, discontinuity in the tangential component of magnetic fields at the interface (surface of periodic graphene ribbons array) between two uniform mediums equals to the conduction current induced on the graphene strips array. The relationship between tangential electric and magnetic fields can be established via the graphene optical conductivity. Through the conventional mode-matching method, the scattering characteristics of the graphene ribbons array can be determined; however, the Gibbs phenomenon in Floquet–Fourier series due to the discontinuity of tangential electric field causes poor numerical convergence in scattering analysis. Specifically, as was reported47, traditional approach of rigorous coupled-wave analysis experienced a poor convergence in particular under the resonance of surface plasmon-polariton wave. Therefore, local basis functions inherently satisfying the field nature in graphene ribbon and slit regions were employed to modify the input–output relation of the periodic graphene ribbons array while the whole formulation can still fit into the standard procedure of Fourier modal method12. Some of the mathematical formulations together with the numerical convergence check -selective property of the SPR curve is essential. A clear dip allows us to easily identify the RI change. In Fig. 50; however, it is apparent to see that the resonant wavelength is varying in accordance with the change in the slit width. Additionally, the line width of SPR curve is getting wide when the slit width is decreasing. This may be explained as the occurrence of strong mutual coupling between two adjacent ribbons for the case of narrow slit width. Moreover, the coupling is caused by the leakage in resonator10, reducing the quality factor (increase in line width) of SPR curve. Contrarily, the SPR curves gradually converges for In Fig. In Fig. The computational design of an evanescent wave biosensor incorporating the SPR on periodic graphene ribbons array is carried out through the electromagnetic modeling and full-wave simulation. Different from the conventional approach by measuring the shift in SPR with respect to the operating wavelength or incident angle of a TM-polarized light, the self-biasing scheme by tuning the chemical potential of graphene (via modifying applied gate voltage) is implemented to track the SPR response. Moreover, due to ultrastrong Supplementary material 1


Retinol isotope dilution (RID) is a well-accepted technique for assessing vitamin A status . Here, in an effort to increase understanding of the method, we briefly review RID equations and discuss their included variables and their coefficients . Then, we focus on contributions of another technique, model-based compartmental analysis and especially the “super-person” approach, that advance the RID method. Specifically, we explain how adding this modeling component, which involves taking 1 additional blood sample from each subject, provides population-specific estimates for the RID coefficients that can be used in the equation instead of values derived from the literature; using model-derived RID coefficients results in improved confidence in predictions of TBS for both a group and its individuals. We note that work is still needed to identify the optimal time for applying RID in different groups and to quantify vitamin A absorption efficiency. Finally, we mention other contributions of modeling, including the use of theoretical data to verify the accuracy of RID predictions and the additional knowledge that model-based compartmental analysis provides about whole-body vitamin A kinetics. Since Furr et al.  publisheIn this article, we delve deeper into RID equations and their included assumptions, with the aim of enhancing understanding of the complexities of the method. Then, we discuss how addition of a super-person modeling component leads to improved confidence in RID predictions by providing population-specific values for the coefficients. We also highlight other ways that model-based compartmental analysis contributes to RID and to our understanding of vitamin A metabolism.Isotope dilution was originally explored by Bausch and Rietz  to estimF is the fraction of a dose of orally administered deuterated vitamin A that is absorbed and retained, dose is the administered tracer dose, S is the ratio of retinol SA in plasma to that in liver after mixing of tracer and tracee, a is a correction for fraction of the dose absorbed and retained but subsequently lost via catabolism, H:D is the postmixing hydrogen-to-deuterium ratio  in plasma retinol, and −1 was described as a correction for the contribution of the dose to vitamin A stores. Furr et al.  to predict liver vitamin A in generally healthy adult surgical patients . Predict\documentF = 0.50 , a = 0.913C10]retinol kinetic data collected in healthy young adults followed for 14 d after ingestion of [13C10]retinyl acetate. The modified equation  to that in stores (SAsit) for individual i at time t. Then:It is informative to rewrite and expand si is the mass of vitamin A in stores for individual iand Msi is assumed to be constant over the duration of the RID study.where Md) either from the literature  or from a super-person compartmental model . TBS predicted for a given subject using derived coefficients are as follows:In practice, mean values used for the coefficients to estimate TBS in an individual are derived .Note that not only SAp) to account for the facts that vitamin A absorption is not 100%, that all of the absorbed dose does not exchange with vitamin A in stores before utilization, and that, because of further vitamin A utilization, not all of the dose that reaches stores is still there at the time of blood sampling; also, to predict the mass of vitamin A in stores by RID, we need S (SApdt/SAsdt) derived either from the literature or from modeling.As implied above, application of an RID equation involves adjusting the measured variable  rather than directly to tissues for utilization before the tracer has mixed with vitamin A in stores. Thus, a better descriptor for F in In addition to absorption efficiency, RID coefficients also need to account for tracer loss between the time of dosing and blood sampling. Along with fecal measurements, cumulative urinary excretion of label was determined over 4–6 d in Sivakumar and Reddy  and Aklaa as used in the Olson and isotope mass balance equations . Because tracer catabolism occurs continually, first following rapid uptake of label from plasma into tissues and later when tracer has mixed with endogenous vitamin A, it makes sense to use a composite term Fa, as in The coefficient quations  assumes S, the ratio of retinol SA in plasma to that in stores, Haskell et al. t is the fraction of the labeled dose in compartment I, Sdt is the ratio of retinol SA in plasma to that in stores, [FaS]dt is the composite RID coefficient, and M(I) is the mass of vitamin A in compartment I; note that values for F(I) and M(I) are provided as part of the model solution. Changes in the coefficients Fa and S as a function of time are shown in Fa, the value peaks at 90% of the absorbed dose at 5 d and then slowly and gradually decreases as catabolism of tracer continues. For S, the value falls to and stays below 1 once SAp crosses over SA in stores (SAs); 1 mo or so after dose ingestion, S will reach and remain at an equilibrium value (Seq) if dietary input of vitamin A is relatively constant. Through theoretical work, we have determined that the value for Seq is R/[R + R], where R   is the rilibrium .FaS]dt. This value can be used to calculate TBS for individuals using each subject's measured SAp at time t ≥4 d. We have shown using data for theoretical subjects that this method provides excellent prediction of TBS for the group  and reasonably accurate values for individuals  . As studies are done in various settings, researchers can determine the CV for FaS at various times and thus recommend when to apply RID, perhaps identifying a universal value for the composite coefficient at the time when the CV% for the composite coefficient FaS is lowest.Although it would be ideal if one could recommend an optimal time for applying RID in children and adults over a wide range of stores, that is not (yet) possible. Present knowledge suggests that, when TBS is low, RID should be done early , but when stores are high, later  is better. From modeling , we knowOverall, the capacity of modeling to provide more accurate values for the RID coefficients helps to justify the additional resources required . If investigators are planning to do a traditional RID experiment in children, it is worth considering the addition of a super-child design, which requires just 1 additional blood sample from each subject, an estimate of vitamin A intake, and a duration of 28–42 d. This will yield not only population-specific values for the RID coefficients, thus improving confidence in the group mean RID estimate of TBS, but it will also allow for reasonably good predictions of TBS in individuals . ImportaFinally, while an RID equation can readily yield a value for TBS, it is impossible to know whether that prediction is accurate—the need for a “gold standard” was highlighted by Quadro . In seve


R2 = 0.744; P < 0.001), with MS demonstrating no loss of PD-L1 protein, even in sections with significant signal loss by IHC impacting diagnostic category. Clones 22C3 and 28-8 were most susceptible to signal loss, with E1L3N demonstrating the most robust signal . Increased humidity and temperature resulted in significant acceleration of immunoreactivity loss, which was mitigated by storage with desiccant. MS demonstrated only modest oxidation of 274 methionine-containing peptides and aligned with IHC results suggesting peptide oxidation is not a major factor. These data imply immunoreactivity loss driven by humidity and temperature results in structural distortion of epitopes rendering them unsuitable for antibody binding following epitope retrieval. Limitations of IHC biomarker analysis from stored tissue sections may be mitigated by cost-effective use of desiccant when appropriate. In some scenarios, complementary MS is a preferred approach for retrospective analyses of archival FFPE tissue collections.Immunohistochemistry (IHC) using formalin-fixed, paraffin embedded (FFPE) tissue is limited by epitope masking, posttranslational modification and immunoreactivity loss that occurs in stored tissue by poorly characterized mechanisms. Conformational epitopes recognized by many programmed-death-ligand-1 (PD-L1) IHC assays are particularly susceptible to degradation and provide an ideal model for understanding signal loss in stored FFPE tissue. Here we assessed 1206 tissue sections to evaluate environmental factors impacting immunoreactivity loss. PD-L1 IHC using four antibodies , raised against intracellular and extracellular epitopes, was assessed in stored FFPE tissue alongside quantitative mass spectrometry (MS). Global proteome analyses were used to assess proteome-wide oxidation across an inventory of 3041 protein groups . PD-L1 quantitation correlated well with IHC expression on unaged sections . Increased humidity and temperature resulted in considerable immunoreactivity loss, particularly among antibodies recognizing extracellular and discontinuous epitopes, which could be partially mitigated with the use of desiccant. However, even in significantly affected tissues MS was able to quantitate PD-L1 expression and assess peptide oxidation. These results suggest MS is suited for biomarker assessments using stored sections—a sample type commonly encountered in oncology research. Formalin-fixation followed by paraffin embedding is the most widely used method for preparing and preserving tissue specimens for clinical and research purposes world-wide. Obtaining, handling, and storing formalin-fixed, paraffin embedded (FFPE) tissue sections presents unique challenges to global pharmaceutical research and development. Standard tissue collections, often influenced by geography and local regulations, can be limiting to the extent that only sectioned tissues adhered to glass slides are available for analysis. This impacts robust biomarker development, since stored FFPE tissue is susceptible to degradation that alters both protein and nucleic-acid assays –5. RecenPrevious studies have found antigen degradation to be both antigen specific and condition dependent; therefore, a detailed understanding of how specific handling and storage conditions affect novel assays warrants greater appreciation in earlier stages of development. FFPE tissue sections are more prone to loss than tissue blocks , 15 and An alternative approach to IHC for protein quantitation in archival tissues is analysis by targeted mass spectrometry (MS) –27. ThisHere we describe a reproducible model of accelerated antigen instability testing and use it to address questions of how immunoreactivity is affected in stored tissue. We specifically evaluate the role of protein oxidation in antigen degradation, how different model IHC assays respond to accelerated conditions, and how MS may be used to evaluate and overcome the limitations of IHC in detecting PD-L1 in stored FFPE tissue sections in the research setting.FFPE tissue blocks of non-small cell lung carcinoma (NSCLC), gastric carcinoma, placenta, and tonsil tissue were commercially acquired from Asterand Bioscience , US Biomax , Tristar Technology Group , and Indiana University Health Methodist Hospital biobank  in either tissue microarray (TMA) or whole section format. Tissue blocks were obtained between 2012 and 2018. Some 1206 tissue sections were evaluated from 35 gastric carcinomas [tubular adenocarcinoma], 10 NSCLCs [2 primary squamous cell carcinoma (SCC) and 8 primary adenocarcinoma (ADC)], 6 tonsil, and 6 placenta samples. Unless otherwise stated, microtomy was performed immediately prior to experiment initiation. Normal storage conditions refer to a monitored and controlled laboratory environment with a relative humidity range of 14.4–80.5% (average 46.8%) and a temperature range of 20.1–31.0 °C (average 21.6 °C) where tissue sections were not exposed to direct light.TM FLEX detection (High pH) on the Autostainer Link 48 (Agilent). Immunostaining for pan-cytokeratin (pan-CK) was assessed as a control using an antihuman Cytokeratin, clone AE1/AE3, ready to use . A representative section from each block was stained for hematoxylin and eosin (H&E) on day 0 of each experiment. All stained tissue sections were scanned on an Aperio ScanScope AT Slide Imager  at ×40 magnification, and images viewed on Aperio ImageScope (v12.3.2)  and humidity levels were measured using a Lockdown Hygrometer . Sections stored within the incubator were removed at appropriate time points for IHC staining alongside control sections stored in normal archival conditions . Repeat experiments of environmental effect on antigen degradation involved the change of these parameters individually. Experiments exploring the use of desiccant to protect against chamber conditions involved comparing antigen expression in sections placed within the chamber in a closed box, sealed in a protective bag  with desiccant  and a humidity indicator card ), to sections placed within the chamber without additional protection, as well as to sections stored under normal archival conditions.13C6, U-15N4-arginine, or U-13C6, U-15N2-lysine at the C-termini and unlabeled peptide standards were purchased from New England Peptide . Isotope labeled peptides were of greater than 99% and 95% isotopic and chemical purity, respectively; absolute concentration was determined by amino acid analysis.Synthetic, isotopically labeled PD-L1 peptide standards LQDAGVYR and AEVIWTSSDHQVLSGK containing U-PD-L1 was analyzed by targeted MS as described previously  with them/z values. Retention times were determined from prior analyses of synthetic peptide standards. The MS1 scan was collected at a resolution of 30,000, an automatic gain control (AGC) target value of 5e4, and a scan range from m/z 350 to 1000. MS1 data were recorded in profile mode. The MS1 scan was followed by targeted MS2 collision induced dissociation scans at a resolution of 30,000, an AGC target value of 5e4, 1.6 m/z isolation window, activation Q of 0.25 and an optimized collision energy for each target of 30%. MS2 data were recorded in profile mode. Parallel reaction monitoring transitions were extracted from raw datafiles and analyzed with Skyline . The mobile phase was initially 5% B and then programmed to 20% B over 18 min, to 35% B over 14 min and finally to 95% B over 5 min before recycling to starting composition. Targeted MS analysis was done by parallel reaction monitoring on the Lumos. The acquisition method consisted of a full scan selected ion monitoring event followed by targeted MS2 scans as triggered by a scheduled inclusion list, with a 5 min retention time window containing the precursor  Skyline . Peptidem/z 375 to 1500. MS1 data were recorded in profile mode. MS2 high-energy collision induced dissociation scans were acquired at an AGC target value of 1e4, a maximum injection time of 35 ms, and with an isolation window of 1.2 m/z. Tandem MS scans were acquired as centroided data. Peptide sequence identification from tandem mass spectra was done as described previously . The mobile phase was initially 6% B and then programmed to 27% B over 27 min, to 40% B over 40 min, and finally to 95% B over 8 min before recycling to starting composition. An MS1 scan was collected at a resolution of 120,000, an AGC target value of 4e5, a maximum injection time of 50 ms, and a scan range from eviously , except eviously . Peptidet test as appropriate. Relationships between variables were assessed using Pearson’s correlation and, if appropriate, linear regression. Analysis of the effect of incubation conditions on methionine oxidized peptides compared the baseline condition and day 28 accelerated degradation condition and used peptide count data for peptides with at least ten spectral counts using Fisher’s exact test (two-sided). The Wilcoxon Signed Rank test was used to determine significance in the number of oxidized peptides between the baseline and day 28 conditions. Analyses of spectral counts were performed using R software for statistical computing (version 3.4.3) and all significances were taken as p < 0.05.Statistical analysis was performed using IBM SPSS statistics software, version 25 . Comparisons of multiple groups over time were performed using repeated measures ANOVA with Bonferroni correction and Tukey’s post-hoc analysis. Comparisons of two groups at a single point were performed using independent samples or paired Student’s p < 0.001), but both clones showed significant loss of PD-L1 expression over time as expected . Examples are shown in Fig. p < 0.05). Clinically relevant loss of CPS (from ≥1% to <1%) was seen by 4.5 months for both clones  with further loss by 24 months .To confirm the PD-L1 immunoreactivity loss in stored tissue sections that has been reported in literature could be reproduced in our laboratory, TMA sections containing 35 gastric carcinomas stored in archive over 24 months under normal ambient conditions were stained for PD-L1 using E1L3N and SP142 clones. Positive pixel count scoring (positivity) demonstrated that E1L3N detected PD-L1 with a greater sensitivity than SP142  and strongly positive (TPS ≥ 50%) cases when assessed with the 22C3 , 28-8 , and E1L3N  clones, but fewer positive and strongly positive cases for SP142 . Loss of PD-L1 TPS in NSCLC sections to levels below these prescribing guideline cutoffs occurred for all clones at varying time points, with over half of cases changing from diagnostically positive to diagnostically negative by day 19 for 22C3 (TPS ≥ 1% and ≥50%), day 9 for 28-8 (TPS ≥ 1% and ≥50%), and day 9 for positive and day 19 for strongly positive for E1L3N  p < 0.001; CK in placenta, 0.626 vs 0.259 (100% vs 41%); p = 0.05, tonsil 0.319 vs 0.219 (100% vs 69%); p < 0.05). Control slides kept at normal ambient conditions had no significant loss of either PD-L1 or pan-CK expression by day 28 for both placenta and tonsil tissue.In order to further understand the relative contribution of major environmental conditions on tissue immunoreactivity, the effect of oxygen, humidity, and temperature on PD-L1 (E1L3N) and pan-CK (AE1/AE3) IHC expression was assessed on tonsil and placenta tissue sections stored in the acceleration chamber set to baseline conditions of 100% oxygen, 37 °C, and 80% humidity over 28 days. By day 28 significant loss of PD-L1 and pan-CK positivity was seen in both tissues (average positivity day 0 vs day 28: PD-L1 in placenta 0.528 vs 0.088 (100% vs 17%); p = 0.174) or pan-CK in both placenta and tonsil tissue ; p = 0.284, tonsil 0.293 vs 0.247 (100% vs 84%); p = 0.423) with a significant reduction seen only for PD-L1 expression in tonsil by day 28 (average positivity 0.135 vs 0.07 (100% vs 52%); p < 0.05) though this was significantly less than the loss seen under 37 °C conditions ; p < 0.05), results summarized in Fig. Changing the temperature in the acceleration chamber had a significant impact on the rate of IHC signal loss. Increasing the temperature to 60 °C (in the context of elevated oxygen and humidity) resulted in extremely rapid loss of PD-L1 expression: . Conversely, decreasing the temperature to 20 °C reduced immunoreactivity loss, resulting in no statistically significant loss of PD-L1 in placenta tissue by day 28 ; p = 0.201) and required 28 weeks for a significant loss in tonsil to occur (0.183 vs 0.021 (100% vs 10%); p < 0.05). The rate of PD-L1 expression loss in reduced humidity conditions was slowed to the extent that both placenta and tonsil tissue demonstrated loss by 39 weeks at 45% humidity similar to, or less than, 1 week at 80% humidity (average positivity for placenta 0.41 vs 0.17 (64% vs 27%); p = 0.13, and tonsil 0.018 vs 0.019 (10% vs 10%); p = 0.93). Average positivity of PD-L1 in placenta and tonsil at 28 days under 45% and 80% humidity are shown in Fig. Reducing humidity had a significant impact on the rate of IHC signal loss. Decreasing humidity to 45% (in the context of elevated oxygen and temperature) resulted in no significant loss of PD-L1 expression in placenta at 39 weeks (0.639 vs 0.408 (100% vs 64%); p = 0.918; tonsil 0.0079 vs 0.016 (7% vs 14%); p = 0.937; pan-CK, placenta 0.43 vs 0.45 (68% vs 71%) p = 0.918; tonsil 0.21 vs 0.22 (61% vs 64%); p = 0.937). Results shown in Supplementary Fig. Reducing oxygen levels in the incubation chamber to 20% (in the context of elevated temperature and humidity) had no significant effect on the rate or total quantity of either PD-L1 or pan-CK expression in either placenta or tonsil tissue ; Because humidity was determined to be a major factor in loss of tissue immunoreactivity, the effect of storing slides with or without desiccant in the acceleration chamber set to baseline conditions of 100% oxygen, 37 °C and 80% humidity over 28 days was examined. This was accomplished by measuring the expression of PD-L1 (E1L3N) and pan-CK IHC using positive pixel count scoring (positivity) on placenta and tonsil tissue sections stored over 28 days.p = 0.083, tonsil 0.088 vs 0.073 (100% vs 83%); p = 0.555, pan-CK in placenta, 0.74 vs 0.72 (100% vs 97%); p = 0.311, tonsil 0.33 vs 0.30 (100% vs 91%); p = 0.185).Upon removal of slides from the sealed container stored within the acceleration chamber, the humidity level was recorded as <30% using the enclosed indicator card. Slides stored with desiccant showed no significant loss of either PD-L1 or pan-CK expression in any tissue at day 28 (Average positivity day 0 vs day 28: PD-L1 in placenta 0.57 vs 0.53 (100% vs 93%); Qualitative assessment of sections showed slides stored with desiccant demonstrate expression loss similar to sections stored under normal atmospheric conditions, with appreciable loss of PD-L1 expression in sections stored without desiccant Figs.  and  5. Tonsil tissue stained for PD-L1 with E1L3N stored in the acceleration chamber with conditions of 100% oxygen, 80% humidity and 37 °C over 28 days was assessed for expression loss difference between crypt epithelium cells and ICs associated with germinal centers. Day 0 sections showed strong staining within the crypt epithelium, and weaker staining within the geminal centers, in keeping with known PD-L1 expression variation between these cell types . Loss isTo test the hypothesis that peptide oxidation is a major contributor to loss of PD-L1 immunoreactivity in stored tissues, targeted MS analyses of PD-L1 peptides were performed to measure PD-L1 protein abundance in specimens stored in normal atmospheric conditions or within the acceleration chamber with conditions of 100% oxygen, 37 °C, and 80% humidity. Two PD-L1 peptides were analyzed, LQDAGVYR and AEVIWTSSDHQVLSGK, both of which are in the extracellular domain of PD-L1. Whereas LQDAGVYR contains no easily oxidizable residues, AEVIWTSSDHQVLSGK contains an oxidizable tryptophan (W) and lies within the recognition sequence for the 22C3 antibody . It has r2 = 0.744, p = 0.001)  Fig. . MS anal01) Fig. .Fig. 6CoOxidation of PD-L1 tryptophan residue in AEVIWTSSDHQVLSGK would occur within the 22C3 recognition sequence and might affect antibody binding. Hydrogen peroxide treatment of the AEVIWTSSDHQVLSGK labeled standard yielded the kynurenine form AEVIW[+4]TSSDHQVLSGK as the predominant product. In MS analyses of the 28 day incubated samples, we detected the AEVIW[+4]TSSDHQVLSGK labeled standard, but not any oxidized peptide from the NSCLC samples . The distributions for ambient stored placenta and tonsil were similar to the 28 day wet oxidized NSCLC from the acceleration chamber . Although these global data indicate the presence of statistically significant oxidation, the distributions clearly indicate a modest overall degree of oxidation that could not account for the near-complete loss of PD-L1 IHC staining observed.To further test the hypothesis that the acceleration chamber produced oxidation of proteins in FFPE sections that interferes with immunoreactivity by IHC, we performed global MS analyses of four samples each of the baseline and day 28 samples from the acceleration chamber experiment, as well as two samples each from placenta and tonsil stored as unstained sections under ambient conditions for 2 years. We extracted data for a set of 274 methionine-containing peptides that were found in all samples in both reduced and oxidized forms and compared abundances as the log2 ratio of spectral counts for both forms (Fig. The loss of immunostaining in archived FFPE tissue is a well-known but poorly understood phenomenon . The rolThe loss of antigen immunoreactivity has been observed to vary between different antigens within and between tissue types, with no obviously consistent factors that may help predict the most sensitive antigens. Both polyclonal and monoclonal antibodies may be affected, and loss occurs in IHC assays that target the nucleus, cytoplasm, or membranes of cells. Specific IHC signal loss can vary between studies depending on factors such as fixation and the specific antibodies used , 37–39.Mechanisms of antigen degradation have been explored previously, with a variety of potential factors thought to influence the loss, including: preanalytical variables, oxidation, humidity, and high temperature , 40, 41.The role of oxidation in antigen degradation has been suggested previously , 40, andPrevious work suggested that humidity or the fixation process can have an effect on antigen degradation , 42–44. The crosslinking process increases accessibility to some antigens and renders others inaccessible through masking of epitopes , 50. TheTissue processing and antigen retrieval prior to IHC involves dehydration and a rehydration of tissue, which has been demonstrated to be a crucial step in achieving successful IHC assay outcomes , 57. IntThis study has limitations. While over a thousand tissue sections were included in the analysis, sample sizes were small for some conditions, perhaps accounting for nonstatistically significant trends in certain experiments. Positivity as defined by pixel counting was complemented by TPS/CPS scores in tumors to give clinical relevance, but the equivalent is not possible in placenta and tonsil; therefore, although significant loss can be quantitatively demonstrated with good reproducibility (Supplementary Figs. To our knowledge, this is the first study that has systematically used quantitative MS to characterize the impact of tissue storage using a model system under controlled conditions. We demonstrate that PD-L1 expression assessed by IHC with different antibody clones undergoes signal loss over time, and that this loss is largely accelerated by humidity and high temperature, rather than by environmental factors favoring oxidation. The use of an acceleration process to mimic the natural loss of antigenicity that occurs in naturally stored FFPE tissue sections may provide a platform by which novel biomarker robustness can be evaluated early in development. MS is a powerful technique that overcomes the limitations of studying protein expression in stored tissues sections by IHC alone. Moreover, MS may be ideally suited to analyze archival FFPE specimens and conduct hypothesis testing in regards to the abundance of protein drug targets with associated therapeutic outcomes.Supplementary figures S1–S9 and supplementary tables S1–S2


Digital assessment is becoming more and more popular within medical education. To analyse the dimensions of this digital trend, we investigated how exam questions (items) are created and designed for use in digital medical assessments in Germany. Thus, we want to explore whether different types of media are used for item creation and if a digital trend in medical assessment can be observed.In a cross-sectional descriptive study, we examined data of 30 German medical faculties stored within a common assessment platform. More precise, 23,008 exams which contained 847,137 items were analysed concerning the exam type  and their respective media content . Out of these, 5252 electronic exams with 12,214 questions were evaluated. The media types per individual question were quantified.The amount of computer- and tablet-based exams were rapidly increasing from 2012 until 2018. Computer- and tablet-based written exams showed with 45 and 66% a higher percentage of exams containing media in comparison to paper-based exams (33%). Analysis on the level of individual questions showed that 90.8% of questions had one single picture. The remaining questions contained either more than one picture (2.9%), video (2.7%), audio (0.2%) or 3.3% of questions had picture as well as video added. The main question types used for items with one picture are TypeA (54%) and Long_Menu (31%). In contrast, questions with video content contain only 11% TypeA questions, whereas Long_Menu is represented by 66%. Nearly all questions containing both picture and video are Long_Menu questions.It can be stated that digital assessment formats are indeed on the raise. Moreover, our data indicates that electronic assessments formats have easier options to embed media items and thus show a higher frequency of media addition. We even identified the usage of different media types in the same question and this innovative item design could be a useful feature for the creation of medical assessments. Moreover, the choice of media type seems to depend on the respective question type. Artificial intelligence, big data, university 4.0, digital transformation and digitalisation are current trends and buzzwords highly discussed in the world of health care and higher education –3. EspecDigitalisation encompasses the emergence of novel tools and applications which offer many new opportunities within the educational sector, especially concerning new assessment formats and content of exams . For thiThe potential addition of media content to questions is the benefit of E-Assessment. In the context of medical education, a study area with the most performed electronic exams , electroThe usage of digital tools on mobile devices allows a layered and dynamic item design, and thus the medical educator has become a designer of items in assessments . The conThe basis for our data evaluation is the assessment platform ItemManagementSystem (IMS) of the Umbrella Consortium for Assessment Networks (UCAN) , 20, wheFor the investigation of the media usage in exam questions, 17,756 paper-based exams were disregarded because paper-based exams offer less possibilities for media addition compared to electronic exams. Thus, the remaining 5252 computer- and tablet-based exams with 205,988 questions were further analysed. For tablet-based exams we distinguish between tablet-based written assessments (tEXAM) and Objective Structured Clinical Examinations (OSCE) evaluated by the examiners via tablet (tOSCE). tOSCE was introduced in 2013 and tEXAM in 2015.TypeA questions are multiple-choice questions with one correct answer.Pick-N questions are also multiple-choice questions, but with more than one correct possible answer.Kprime questions are basically yes or no questions where each possible answer must be marked as true or not true.Freetext questions have a textbox where any desired answer can be given.Long_Menu question have usually a long answer list  to choose from and thus are comparable to freetext questions where the knowledge of the right answer is presumed.OSCE is an assessment format for skill testing in written or tablet-based form that was first described by Harden et al. , 22. In First, the different media types per single question were quantified and second the preferred question type for each media type determined. We categorised the questions into six different classes: TypeA, Pick-N, Kprime, Freetext, Long_Menu and OSCE:The evaluation of the compiled data occurred through the software packages Microsoft Excel . With the Excel software mean values and standard deviations were determined. The dependency of question type and media type was analysed with R  using Pearson’s Chi-squared test.Figure The average percentage of exams containing media with reference to all exams of the respective exams type is illustrated in Fig. In the investigations what kind of media types are used in electronic exams, it is not surprising that pictures are still the main media type used in combination with text Fig. . AdditioTo analyse the used media type in E-Assessment, 5252 electronic exams were evaluated with 2065 exams that contained either picture, video or audio as media types. These exams contained 12,214 questions that were further assessed individually regarding their added media types. In accordance with the above-mentioned data, the evaluation of questions showed that pictures are indeed the main media type in combination with the question text .We investigated whether a particular media type is favoured for a special question type. Our findings show that 54% of the questions containing one picture can be assigned to single answer multiple choice questions (TypeA) (Fig. The current study shows the digital trend towards electronic exams in German medical schools. The quantity of electronic assessment methods, such as computer- and tablet-based exams has grown strongly until the end of 2018 according to our analysis. The frequent usage of picture items could be demonstrated with our data since pictures still represent the main media type used in medical assessments. Our data shows that other media types such as audio and video are quite rarely used within medical education in Germany. Perhaps these media types are more advantageous for other areas of studies. Within the musicology, parts of musical works could be played and learning foreign languages native speakers could be listened to which is already an element of learning management systems –25 and pIn general, good quality questions used for exams are costly  and one Unfortunately, the authors did not find any references regarding nationwide studies to determine a change in media usage after implementation of digital exams. However, there is a trend in the use of digital assessment formats worldwide –32. As eThere are some limitations to our study. First, we only analysed the data of one specific database which depends on the accurate entering of the data by its users. Second, we have only focused on medicine as a subject of study and only considered German medical faculties. Third, our exam exclusion criteria may have resulted in exclusion of potentially relevant exams or questions. Moreover, the number of some question type and media type combination in our analysis might be rather low for less frequently used question types. Nonetheless, this study is the first attempt to compare and analyse the used media types for medical exam questions in different exam types.Further research could incorporate the analysis whether the use of media in exam questions is beneficial for the quality of the assessments and thus the quality of medical study. For that reason, statistical data like the difficulty, discriminatory power and reliability of exam questions with and without media should be collected. An automatic upload of statistical values does currently not take place in the examined assessment platform, but is planned for the current year, which is why a statistical analysis of exam questions would be feasible in the future and is aimed at. Furthermore, it should also be explored to what extent different medical competencies and knowledge can be tested depending on the chosen media and question type. Hurtubise and colleagues discussed the usage of video clips linked to different competency domains . In GermIn the age of digitalisation, a digital trend is occurring in higher education in Germany. In our study we analysed how this digital change is affecting assessments in medical education. Interestingly, an increased number of media additions like picture, video and audio can be observed in electronic exams and even media combinations of text, picture and video are utilised for the creation of exam questions. So far, the usage of different media types in the same question is quite rare, but modern digital technologies together with the collective exchange of items in a common item-pool and a broad range of available assessment tools will facilitate these innovative digital assessment formats in the future. In addition, our study demonstrates that some media types are more used for a specific question type than others. Thus, one could speculate that our data shows that specific media types are more suitable for some question types than for others.