Neurofascin mRNA, encoding the cell adhesion molecule essential for paranode assembly and maintenance. Loss of TDP-43 triggers the retention of a previously unidentified cryptic exon, which targets Neurofascin mRNA for nonsense-mediated decay. Thus, TDP-43 is required for neurofascin expression, proper assembly and maintenance of paranodes, and rapid saltatory conduction. Our findings provide a framework and mechanism for how Schwann cell-autonomous dysfunction in nerve conduction is directly caused by TDP-43 loss-of-function.TDP-43 is extensively studied in neurons in physiological and pathological contexts. However, emerging evidence indicates that glial cells are also reliant on TDP-43 function. We demonstrate that deletion of TDP-43 in Schwann cells results in a dramatic delay in peripheral nerve conduction causing significant motor deficits in mice, which is directly attributed to the absence of paranodal axoglial junctions. By contrast, paranodes in the central nervous system are unaltered in oligodendrocytes lacking TDP-43. Mechanistically, TDP-43 binds directly to  Tardbp) is a nucleic acid-binding protein that regulates the processing of a wide range of coding and noncoding RNAs by directly binding to transcripts and/or interacting with key RNA-processing complexes , which encodes a glial cell adhesion molecule necessary for paranodal junction formation and maintenance, by repressing the usage of a cryptic exon during splicing. Our findings are the first demonstration of a functional role for TDP-43 in axon–glial interactions in the PNS and that the loss of function for TDP-43 in Schwann cells results in impaired conduction velocity and motor behavior.Not surprisingly, the function of TDP-43 is mainly focused in neurons . Howeverfl/flTardbp)   with Dhh channels and their associated protein complex from juxtaparanodal regions to paranodes  do not exhibit a gross neurological deficit when observed in their home cage. However, when challenged with the rotarod test, the cKO mice perform significantly worse than WT littermates . Intriguingly, when the sequencing reads are aligned to the Nfasc locus and analyzed, we find reads mapped to introns 17 and 18 of Nfasc. Because intron 17 contains a UG-rich sequence, a known consensus RNA-binding motif for TDP-43 . At 3 months post-tamoxifen injection, NFasc is absent from a similar proportion of Schmidt–Lanterman incisures in the sciatic nerves and ventral roots  . While Nnn cells . The pronn cells , suggestNfasc gene, which overlaps with TDP-43-binding sites. Schwann cell NFasc protein is known to be essential for paranodal junction assembly and maintenance  is highly enriched at the axon initial segments and nodes and is required for maintaining the high density of Nav channels therein  (Cnp-Cre (-/-Cntnap1 (Caspr KO) (Mpz-CreERT2 (P0-CreERT2)    ; Mpz-CreCreERT2) . All expThe following primary antibodies were used: mouse anti-actin , mouse anti-AnkB , mouse anti-AnkG , mouse anti-AnkG , mouse anti-βII spectrin , chicken anti-βIV spectrin (a gift from Dr. Matthew N. Rasband), rabbit anti-βIV spectrin , mouse anti-Caspr (Mab275) , rabbit The sciatic nerves, trigeminal nerves, and spinal cords were either dissected after transcardial perfusion and postfixed as described previously  or freshAfter air dried, the tissues on the coverslips were blocked with 10% (v/v) normal goat or donkey serum, 0.3% (v/v) Triton X-100 in PBS for 30 min, and stained with primary antibodies diluted in the blocking solution at 4°C overnight. After one wash with the blocking solution and two washes with PBS for 5 min each, the tissues were stained with secondary antibodies diluted in the blocking solution at room temperature for 1 hr. After one wash with the blocking solution and two washes with PBS, the coverslips were mounted onto glass slides in Vectashield Vibrance mountant (Vector Laboratories) or ProLong Diamond mountant (Thermo Fisher Scientific).Nerve conduction studies were performed as described previously with modification . In brieAfter sacrificing the mice, the sciatic nerves were exposed and the lower bodies of the mice were fixed in 4% (w/v) PFA, 2.5% (w/v) glutaraldehyde in 0.1 M sodium cacodylate, 5 mM calcium chloride, pH 7.4 at room temperature overnight, and at 4°C for 2 days. Nerves were dissected and stored in the same fixative at 4°C for at least 2 days. Nerves were then processed as previously described  and wereFour-week-old mice were first familiarized with the rotarod (Ugo Basile 47600 V04) at 4 rpm for 5 min. Thirty minutes later, the mice were challenged with accelerating rotarod from 4 to 40 rpm within 5 min for three trials, separated by 30 min. The next day, the mice were tested with the rotation speed increased from 4 rpm to 24 or 32 rpm within 30 s and maintained at 24 or 32 rpm, respectively, for another 60 s. Three trials were performed for each speed, and the mice were allowed to rest for at least 10 min between trials. The time periods for which each mouse remained on the rotarod at each speed were recorded and averaged.Sciatic nerves were homogenized with sonication in 320 mM sucrose, 5 mM sodium phosphate (pH 7.2), 0.2 mM sodium fluoride, 0.2 mM sodium orthovanadate, 1× cOmplete protease inhibitor cocktail (Roche), and 1 mM phenylmethylsulfonyl fluoride on ice. The protein concentration was determined with the BCA protein assay kit (Thermo Fisher Scientific) using the standard curve derived from a serial dilution of bovine serum albumin; 30 μg of total protein was subjected to western blotting.g for 15 min at 4°C. The total RNA was purified from the supernatant using the Direct-zol RNA Miniprep kit (Zymo Research) by following the manufacturer’s instruction including the in-column DNase I treatment. 0.5 μg of total RNA (concentration determined by the absorbance at 260 nm) was used in 20 μl of reverse transcription reaction with LunaScript RT SuperMix (New England Biolabs) and its No-RT Control Mix according to the manufacturer’s instruction. The RT and No-RT reactions were then diluted to 1/4× with deionized water, and 2 μl was used for qPCR in 25 μl of reaction containing 0.5 μM primers with Power SYBR Green PCR Master Mix and QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). The thermocycling parameters are 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min, followed by melting curve analysis. The primers used in qPCR are as follows (from 5′ to 3′): Tardbp ex3 , Tardbp ex4-5 , pan Nfasc , NF155 , NF140+ex27 , NF140-ex27 , G6pdx , and Polr2a . Polr2a was chosen as the reference gene . The efficiency of each primer pair was validated to be between 90% and 110% with six points of threefold or fourfold serial dilutions of P16 mouse sciatic nerve cDNAs or P31 mouse spinal cord cDNAs. The primer specificity was screened on the Ensembl genome browser by using the BLASTN search tool against the cDNA database and genomic sequence, and confirmed by gel electrophoresis and sequencing from both ends of the PCR products. Technical triplicates of the RT reaction and duplicates of the No-RT reaction were performed for each biological replicate, and three biological replicates were analyzed. The relative transcript level was calculated by the ΔΔCq method. Comparison of cHet or cKO with WT was carried out by one-sample unpaired two-tailed t-tests with the test mean equal to 1.Sciatic nerves were homogenized in the TRIzol reagent (Thermo Fisher Scientific) using a Dounce homogenizer (Wheaton) and incubated at room temperature for 5 min. The homogenate was extracted with chloroform, incubated at room temperature for 3 min, and centrifuged at 12,000×nce gene , and theNfasc gene spanning exons 17 and 20 were (from 5′ to 3′): GCAAAGGCCTACCTCACTGT (forward) and CTCGTTGACAGCGATGACTC (reverse). Similar results were also obtained by using GACCAGGGCAGTTACACG  and TCACACTCCTCTCAGCCAG . The PCR products were resolved in agarose gel.Total RNA was extracted from sciatic nerves and spinal cords using the TRIzol reagent according to manufacturer’s instruction. After DNase treatment using RQ1 RNase-Free DNase (Promega), 0.5–1 μg of RNA was reverse transcribed using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific) and subsequently used for PCR. Primers for mouse fl/flTardbp, fl/+Cnp-Cre;Tardbp, and fl/flCnp-Cre;Tardbp mice (n = 3 per genotype) were used to prepare RNA-seq libraries using the TruSeq RNA Sample Prep Kit (Illumina). Illumina HiSeq4000 was used for paired-end 151 bp sequencing and yielded 30–40 million reads per sample. This data has been deposited in NCBI's Gene Expression Omnibus . The data points of five or less were assumed to be drawn from normal distributions. For the ANOVA tests, homogeneity of variance was not rejected by Levene’s tests. Biological replication is achieved by analyzing different mice. Technical replication is achieved by repeating the experiments using the same samples. The number of mice or technical replicates used for each comparison is described in the figure legends and Materials and methods. No data points were excluded except that one WT mouse in the rotarod test was considered an outlier, which may have learned that the consequences of falling are innocuous  is reduced, while the neuronal splice form encoding NF186 is unaltered. While it is likely that TDP-43 affects additional transcripts in Schwann cells, this study reveals one key protein that is altered as a consequence of TDP-43 deletion. As loss of TDP-43 function is associated with numerous disease and injury states, the studies have important implications for the consequences of these changes in Schwann cells.During development, selective deletion of TDP-43 from Schwann cells revealed a cell-autonomous function in paranodal junction assembly and motor nerve conduction, but not formation of compact myelin. Mechanistic studies identified that the production of the glia-specific splice form of Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Gary Westbrook as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Roman Giger (Reviewer #1); James Salzer (Reviewer #2); Phil Wong (Reviewer #3). The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Thank you for submitting your article "TDP-43 maximizes nerve conduction velocity by repressing a cryptic exon for paranodal junction assembly in Schwann cells" for consideration by eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:Nfasc mRNA  results in a premature stop codon, triggering nonsense-mediated decay of its mRNA. The absence of this protein provides an explanation for the altered assembly of paranodal junctions, the marked delay in peripheral nerve conduction and associated motor discoordination. Nuclear exclusion of TDP-43 occurs in many cell types following injury and progressive disease, raising the possibility that Schwann cell dysfunction may also contribute to pathological changes during these conditions.This study describes the consequences of selective deletion of the of the RNA binding protein TDP-43 from Schwann cells. Using in vivo genetic manipulations in mice, the authors show that TDP-43 is required for both the formation and maintenance of paranodal junctions in the peripheral nervous system. TDP-43 is known to suppress the incorporation of cryptic exons in diverse mRNAs. In Schwann cells lacking TDP-43 they find that inclusion of a cryptic exon in Essential revisions:1) Provide information about the characteristics of axons and myelin in the Tardbp cko mice . Changes in these features could also affect conduction velocities and contribute to paranodal defects. This is particularly important, given the large number of potential mRNA targets of TDP-43 and the observation that myelination is delayed in the conditional mutants, a phenomenon not normally observed in paranodal mutants. If this analysis is not possible in a reasonable time frame, please describe in detail the observations that can be made from existing electron micrographs and revise the text to discuss how alterations in other aspects of these myelinated nerves could contribute to the phenotypes exhibited by the Tardbp cKO mice.2) Revise Figure 1. It is difficult to appreciate that TDP-43 is present in normal Schwann cells without displaying the Sox10 channel alone. Quantification of overlap should be provided, along with evidence that nuclear TDP-43 in non-Schwann cells was not affected by this manipulation. In Figure 1A it appears that Sox10 is strongly upregulated in cKO nerves. If this is the case, provide quantification, if not, replace figure panel with a more representative example (or single channel images). Dhh-Cre is active in endoneurial fibroblast precursors , so it is unclear what cell type persistently express TDP43 in the P28 Dhh-Cre cKOs. The quality of the images of isolated nerves in panel D could be improved.3) Revise the discussion of TDP-43 splicing repression in human Schwann cells. The lack of conservation between cryptic exons between species make this hypothesis very speculative. It would enhance the impact of the study to search available databases for evidence in humans of TDP-43 targets related to paranodal junction genes. Such human data would provide strong clinical implications of findings from this manuscript. It is possible that other TDP-43 targets related to paranodal junction are impacted in ALS patients. However, inclusion of these data is not deemed necessary for publication.4) Revise Figure 2. Figure 2H: In assessing behavioral outcomes of TDP-43 cKO mice, a mild defect in rotarod test was observed and it appeared that there is a bimodal distribution where one group of mice show a deficit whereas the other group seemed normal. Can this be explained by extent of neurofascin cryptic exon inclusion or gender of mice? Is there assessment using an independent behavioral task to confirm this finding? Given the non-normal distribution, a standard t-test is not appropriate for this statistical comparison. Essential revisions:1) Provide information about the characteristics of axons and myelin in the Tardbp cko mice . Changes in these features could also affect conduction velocities and contribute to paranodal defects. This is particularly important, given the large number of potential mRNA targets of TDP-43 and the observation that myelination is delayed in the conditional mutants, a phenomenon not normally observed in paranodal mutants. If this analysis is not possible in a reasonable time frame, please describe in detail the observations that can be made from existing electron micrographs and revise the text to discuss how alterations in other aspects of these myelinated nerves could contribute to the phenotypes exhibited by the Tardbp cKO mice.t test). The myelin thickness of the WT and cKO is comparable and does not contribute to the conduction delay along the cKO nerves. We find that the myelinated axons in the cKO tend to have smaller diameters; however, because the absence of paranodal junctions is known to cause ~40-50% conduction delay , and we observed a 52% delay in the TDP-43 cKO, we believe that the smaller axon diameter in the cKO only contributes to a small portion in the delay of nerve conduction. We have revised the manuscript to discuss this variable.We want to thank the reviewer for this important comment, as it constitutes an alternative conclusion. We have now measured the g-ratios and axon diameters from sciatic nerves of three WT and three cKO mice at P21, and the data is now presented in Figure 1—figure supplement 2. The g-ratios between the WT and cKO mice are not statistically different (p = 0.1214 by 2) Revise Figure 1. It is difficult to appreciate that TDP-43 is present in normal Schwann cells without displaying the Sox10 channel alone. Quantification of overlap should be provided, along with evidence that nuclear TDP-43 in non-Schwann cells was not affected by this manipulation. In Figure 1A it appears that Sox10 is strongly upregulated in cKO nerves. If this is the case, provide quantification, if not, replace figure panel with a more representative example (or single channel images). Dhh-Cre is active in endoneurial fibroblast precursors , so it is unclear what cell type persistently express TDP43 in the P28 Dhh-Cre cKOs. The quality of the images of isolated nerves in panel D could be improved.We now include the single-channel images for TDP-43 and Sox10 from P28 in Figure 1—figure supplement 1, together with those from P3. All the Sox10-positive nuclei are TDP-43-positive in the WT. The intensity of Sox10 staining varies among different nuclei of the WT and cKO especially at P28, and we did not observe a change in the Sox10 levels in the cKO.We thank the reviewers for bringing our attention to the study by Joseph et al., 2004. We observed the complete loss of paranodal junctions in the PNS using three different Cre driver lines to conditionally knock out TDP-43 in our study—Dhh-Cre, P0-Cre and Cnp-Cre—and this strongly indicates that the phenotype we observe is cell-autonomous to Schwann cells. Furthermore, we have stained the endoneurial fibroblasts with PDGFRα and confirmed that the fibroblasts are among the TDP-43-positive/Sox10-negative population in the cKO mice (by Dhh-Cre), which includes other PDGFRα-negative, non-fibroblast cells as well . The apparent discrepancy between our observation and that of Joseph et al. with the Dhh-Cre in endoneurial fibroblasts may be attributed to the different floxed alleles involved. This has been shown to be the case for Cnp-Cre . In brief, when combining the Tardbp floxed allele and Dhh-Cre, we do not observe recombination in endoneurial fibroblasts.We have changed panel D of Figure 1 to improve the picture quality.3) Revise the discussion of TDP-43 splicing repression in human Schwann cells. The lack of conservation between cryptic exons between species make this hypothesis very speculative. It would enhance the impact of the study to search available databases for evidence in humans of TDP-43 targets related to paranodal junction genes. Such human data would provide strong clinical implications of findings from this manuscript. It is possible that other TDP-43 targets related to paranodal junction are impacted in ALS patients. However, inclusion of these data is not deemed necessary for publication.NFASC pre-mRNA  Revise Figure 2. Figure 2H: In assessing behavioral outcomes of TDP-43 cKO mice, a mild defect in rotarod test was observed and it appeared that there is a bimodal distribution where one group of mice show a deficit whereas the other group seemed normal. Can this be explained by extent of neurofascin cryptic exon inclusion or gender of mice? Is there assessment using an independent behavioral task to confirm this finding? Given the non-normal distribution, a standard t-test is not appropriate for this statistical comparison.The complete loss of paranodal junctions has been confirmed in at least two cKO mice per Cre driver line , and the paranodal phenotype has a 100% penetrance. We have included more mice in the rotarod analysis for a total of 10 mice per genotype. The cKO data points are now more evenly distributed , and the normality of the data point distribution is not rejected by the Shapiro-Wilk test (p = 0.1513 for 24 rpm and p = 0.1949 for 32 rpm). The majority of the WT mice could remain on the rotating rod until the cutoff time of 90 sec and indeed the normality of the WT distribution is rejected by the Shapiro-Wilk test. Therefore, we have switched to the Mann-Whitney statistical analysis and now present the data in box-and-whisker plots. The motor deficit of the cKO mice is now confirmed with a larger group of mice and by using the Mann-Whitney test (p = 0.0115 for 24 rpm and p = 0.0036 for 32 rpm). We have revised the manuscript and source data file accordingly.


AlGaN/GaN fin-shaped field-effect transistors (finFETs) with a graphene gate were fabricated and studied. These devices demonstrated ~8 order of magnitude on/off ratio, subthreshold slope of ~1.3, and low subthreshold current in the sub-picoamperes range. The effective trap density responsible for the 1/f low-frequency noise was found within the range of (1–5) · 1019 eV−1 cm−3. These values are of the same order of magnitude as reported earlier and in AlGaN/GaN transistors with Ni/Au Schottky gate studied as a reference in the current study. A good quality of graphene/AlGaN Schottky barrier diodes and AlGaN/GaN transistors opens the way for transparent GaN-based electronics and GaN-based devices exploring vertical electron transport in graphene.Electrical and noise properties of graphene contacts to AlGaN/GaN heterostructures were studied experimentally. It was found that graphene on AlGaN forms a high-quality Schottky barrier with the barrier height dependent on the bias. The apparent barrier heights for this kind of Schottky diode were found to be relatively high, varying within the range of φ The invention of graphene opened the road to new exciting physics and new devices based on the unique ultimate geometry of graphene and its specific physical properties and effects. Among them are extremely high carrier mobility ,2,3, quaThe majority of the graphene research deals with in-plane  current transport. A much less explored field is vertical transport through graphene and other two-dimensional materials. Indeed, there are just a few fields of research activity in this direction: transparent electrodes for light-emitting diodes and displays , verticaAnother promising field, which is the topic of the current letter, is graphene electrodes for electronic devices. It is known that graphene can form either an ohmic or Schottky barrier contact to semiconductors (see Reference  and refeThe combination of graphene on the top of the AlGaN layer in AlGaN/GaN high-electron-mobility transistors (HEMTs) and 2D electron gas (2DEG) on AlGaN/GaN interface create a unique system with two closely spaced high-conductivity 2D layers. This kind of system is a promising platform to study the Coulomb interactions  and two-Although graphene/AlGaN/GaN heterostructures are promising for several applications, the properties of them are only preliminary studied.In this work, we studied the properties of the graphene for AlGaN contact and the behavior of AlGaN/GaN finFETs with the graphene Schottky barrier gate. Special attention was paid to the low-frequency noise properties, which characterize the quality of the structures and are crucial for the majority of applications.0.15Ga0.85N barrier layer, 2.7 μm intentionally undoped GaN layers, and 100 nm Al0.03Ga0.97N buffer. The schematic of the layer sequence and qualitative band diagram of the structure are shown in The AlGaN/GaN structures were grown by the Metalorganic Vapour Phase Epitaxy method in the closed coupled showerhead 3 · 2 inch Aixtron reactor . The epistructure consisted of 2 nm GaN cap, 25 nm AlThe device processing was performed using a commercial laser writer system  for lithography processing with the 405 nm GaN laser source. In order to separate devices on the wafer, the shallow 150 nm mesas were etched using Inductively Coupled Plasma-Reactive Ion Etching (ICP-RIE)    starting from the edge of the stack. As a result, Gr/PMMA separated from Cu foil and remained floating on the electrolyte surface. After that, the detached Gr/PMMA stack was moved to deionized water to rinse the electrolyte residue, transferred onto AlGaN/GaN structure, and heated at ~130 °C to make better contact. Finally, the PMMA support was carefully removed in acetone, and the structure with graphene was rinsed in IPA. This procedure allowed us graphene deposition on large area substrates of 1 inch or more in diameter. Measurements of the back-gate transistors fabricated using this graphene indicated its p-type. We used a commercially available single-layer graphene  produced by chemical vapor deposition (CVD) method on the surface of thin copper foils . The ste−1), 2D (2680 cm−1), and D (1350 cm−1). Low Full Width at Half Maximum (FWHM about 32 cm−1) of the 2D peak, as well as a much higher intensity of the 2D mode by comparison with the intensity of the G peak (2D/G > 2) are characteristic of the monolayer of graphene. Very low D mode amplitude indicates the high quality of the graphene structures. After high-speed electrochemical delamination, graphene was patterned in oxygen plasma using an Oxford Instruments Plasmalab 80 Plus ICP-RIE system .The quality of the graphene layers was controlled with Raman spectroscopy and Scanning Electron Microscopy (SEM). The surface morphology of the transferred graphene samples was investigated by Auriga CrossBeam Workstation (Carl Zeiss) . As a result of the etching, graphene remained on the mesas leaving the short ends of the rectangular mesas free from it. These parts of the mesas were used to fabricate ohmic contacts to 2DEG. In order to form ohmic contacts, Ti/Al/Ni/Au (150/1000/400/500 Å) layers were deposited and rapid thermal annealed at 800 °C in the nitrogen atmosphere for 60 s. Parts of the graphene remained out of the mesas on GaN buffer and were used to fabricate Cr/Au (50/1850 Å) ohmic contacts to it. The resulting design represents finFET with the graphene gate. This design allowed us to study the properties of the graphene AlGaN Schottky barrier and the characteristics of the finFETs themselves.g = 5–20 µm and Wg = 4–20 µm, respectively. As a reference, finFETs with standard Ni/Au (250/750 Å) Schottky barrier gates were also fabricated.The schematic view of the fabricated finFET and optical microscope image of one of the graphene gate transistors are shown in v is the drain voltage fluctuations and Rd is the differential device resistance.All measurements were done at room temperature using a probe station. The spectral noise density of drain current fluctuations was calculated as g). In accordance with the thermionic emission (TE) model, the current–voltage characteristic of the Schottky diode at B is the Boltzmann constant, T is the temperature, A* is the modified Richardson constant, S is the contact area, and φb is the barrier height. Although under the TE model the ideality factor n = 1, experimental characteristics are always characterized by n > 1. The higher than unity ideality factor can be attributed to the effects of image force barrier lowering and the non-idealities of the barrier, such as inhomogeneity of the barrier height and the contribution of other current mechanisms . Therefore, the ideality factor is often used as a figure of merit of the barrier quality. The current-voltage characteristic of Au/Ni/AlGaN Schottky barrier diode in As seen, the Au/Ni/AlGaN Schottky barrier diode demonstrates exponential forward current–voltage characteristics I–exp  eV for all studied diodes. The apparent barrier heights for the graphene/AlGaN diode in b = (1.0–1.26) eV.Equation (1) allows us to estimate the saturation current and the barrier height. However, the ideality factor n > 1 is an indication of the non-ideality of the barrier. Therefore, the barrier height for the diodes with the ideality factor significantly higher than unity should be considered only as an apparent barrier height. We found φThe barrier height for Ni/AlGaN Schottky barrier reported in the publications is within the range of (0.85–0.99) eV  at frequency f = 10 Hz for three representative graphene gate transistors are shown in The dependences of the relative spectral noise density t is the effective trap density, s is the concentration, and γ is the attenuation coefficient of the electron wave function under the barrier:In the McWhorter model, the spectral noise density of the drain current fluctuations given by :(2)SII2=8 cm−1. For the AlGaN/GaN system, the attenuation coefficient is somewhat smaller: γ ≅ 0.45 × 108 cm−1 [Here m* is the electron effective mass, h is the Planck’s constant, and Φ is the tunneling barrier height at the interface. In Si MOSFETs, the γ value is usually taken to be γ = 10108 cm−1 .s ~ (Vg − Vt), the McWhorter model predicts the dependence of noise on the gate voltage as t on energy (gate voltage). As seen in Since in the linear regime nferences ,25). Them is the external transconductance, q is the electron charge, and C is the gate capacitance per unit area. This method allows us to get rid of the uncertainty of the threshold voltage and take into account the possible influence of the contact resistance [The effective trap density can be also found from the input gate voltage noisesistance .g. This effect can be due to the dependence of the trap density on the energy. At high gate voltage, the trap density is close to 1019 eV−1 cm−3. Similar or higher values of the effective trap density within the range of (1018–1020) eV−1 cm−3 were found in the majority of publications on Si MOSFETs with a high-k dielectric (see References [t ≅ 1017 eV−1 cm−3 [t = (1018–1020) eV−1 cm−3 (see t = (2.3 × 1019–1.7 × 1020) eV−1 cm−3 in AlGaN/GaN HEMT structures with Ni/Au gate.One can see that the effective trap density for studied devices only weakly depends on the gate voltage, reflecting the ferences ,29,30,312O or O2 plasma oxidation treatment of AlGaN surface strongly increases the noise. On the other hand, AlGaN/GaN transistors with insulated Al2O3 or SiO2 gate have shown to have lower noise [Although AlGaN/Schottky barrier interface is relatively far from the two-dimensional channel, it is known that this interface still may affect the noise originated from the channel. In Reference , it was er noise . Therefob = (1.0–1.26) eV. The relatively high value of the apparent barrier height in comparison with other published results is an indication of low defect concentration in AlGaN and good quality of the interface. AlGaN/GaN finFETs demonstrated good characteristics with ~8 order of magnitude on/off ratio and subthreshold slope of ~1.3. The effective trap density found from the low-frequency noise measurements was ≥1019 eV−1 cm−3, which is of the same order of magnitude as in the transistors with Ni/Au Schottky gate. This is an indication that graphene forms a high-quality Schottky barrier to AlGaN which can serve as a gate for various types of transistors. It is important that the open surface of graphene does not deteriorate electrical and noise properties of 2D gas on the AlGaN/GaN interface. These results pave the way towards transparent high-temperature GaN-based electronics and to study Coulomb interactions in the graphene/AlGaN/GaN system.In summary, we fabricated and studied graphene gate AlGaN/GaN finFETs. Study of the vertical transport in graphene/AlGaN Schottky diodes indicated the dependence of the Schottky barrier on the bias. The apparent barrier heights for the graphene/AlGaN diodes were found to be within the range of φ


Giant cell myocarditis (GCM) is a rare and rapidly fatal cardiovascular condition most often seen in young adults. It is characterized microscopically by myocardial necrosis with multinucleated giant cells in the absence of well-defined granulomas. This disorder has typically been attributed to manifest as heart failure, but in some individuals, GCM may present as sudden cardiac death. Herein, we present a fatal case of GCM in a 36-year-old male with a history of autoimmune disorders. The decedent presented to the emergency room due to vomiting and was treated for nausea due to suspected dehydration. He was discharged that night and found dead on his bathroom floor the following day. Postmortem examination revealed psoriasis and granulomatous lesions in the lungs consistent with sarcoidosis, further supporting circumstantial evidence existing between GCM and autoimmune disorders. Additionally, this case provides an opportunity to distinguish GCM from the distinct clinical entity of cardiac sarcoidosis (CS), especially in the setting of systemic sarcoidosis. We hope to raise awareness of this rare disease process and its potential to cause sudden cardiac death so that it may be considered in a differential diagnosis as immunosuppression and early cardiac transplantation largely determine the prognosis. Giant cell myocarditis (GCM), commonly referred to as idiopathic giant cell myocarditis (IGCM), is a rare disorder that typically affects young to middle-aged adults that was first described in 1905 as a mixed myocardial infiltrate with multinucleated giant cells and cardiomyocyte necrosis.,Giant cell myocarditis is extremely rare with less than 300 reported cases since its initial distinction from myocarditis in 1905,Herein, we present a case of SCD due to GCM in a patient also found to have probable pulmonary sarcoidosis with the goal of increasing our understanding of the defining characteristics, presentation, distinction, and etiology of GCM along with exploring the relationship of GCM with sarcoidosis and other autoimmune diseases.A 36-year-old white male who reported not feeling well was seen at the local emergency room for nausea and vomiting due to suspected dehydration. He was given intravenous fluids, ondansetron, and metoclopramide and then deemed stable for discharge. He was also prescribed promethazine for nausea and was instructed to continue taking his home medications, which included halobetasol, lisdexamfetamine, and sertraline. He was found unresponsive on his bathroom floor by his mother the following afternoon and death was pronounced that evening. The autopsy was performed the following morning. Access to previous encounters was not available.The decedent’s external examination was largely unremarkable, showing a well-developed, well-nourished, adult Caucasian male who weighed 75.8 kilograms and was 1.70 meters in height. Pink, scaly skin covered the extensor surfaces of his upper and lower extremities, consistent with his clinical history of psoriasis.The heart weighed 510 grams  and was otherwise unremarkable externally. Upon sectioning, the myocardium was dark, red-brown, and firm with multiple areas of yellow-tan discoloration involving both ventricles and extending from apex to base . These aThree sections were taken for microscopic examination, and revealed geographic replacement of myocardium by confluent areas of central fibrosis and necrosis with scattered multinucleate giant cells and a leading edge with a mixed inflammatory infiltrate with lymphocytes, plasma cells, eosinophils, and giant cells with associated myocyte necrosis 22C. AddiThe right and left lungs weighed 790 and 830 grams respectively  and were found to be edematous grossly. The upper and lower airways were patent, smooth, yellow-tan and largely unremarkable. There were notable focal dense fibrous adhesions of the left lung to the lateral chest wall adjacent to an old surgical site.Three microscopic sections were submitted and showed scattered, well-defined, non-caseating granulomas with minimal associated inflammation throughout the parenchyma, some containing prominent asteroid bodies .These granulomas were present within the interstitium and were both peribronchial and perivascular in distribution and consistent with pulmonary sarcoidosis. Bilateral hilar lymphadenopathy was appreciated.Other pathologies considered in the differential diagnosis for the pulmonary granulomas included mycobacterium tuberculosis, histoplasmosis, blastomycosis, bronchocentric granulomatosis, hypersensitivity pneumonitis, pneumoconiosis, foreign body granulomatosis, granulomatosis with polyangiitis, and eosinophilic granulomatosis with polyangiitis, but, no history of recent travel to endemic areas, environmental or occupational exposures, prior illnesses, IV drug use, or evidence of vasculitides make these etiologies less likely. The patient’s lack of systemic symptoms such as fever, night sweats, and weight loss also decreased concern for infection or malignancy. No foreign material was identified under polarized light and specialized stains for mycobacterium and fungal organisms were not deemed necessary. Hypersensitivity pneumonitis (HP) was given additional consideration, but HP granulomas are typically smaller, not discrete, and are associated with greater interstitial chronic inflammation. These characteristics were inconsistent with our findings.Gross examination of the other organs was unremarkable. The liver was enlarged and weighed 2240 grams , but there was no significant edema or cavity effusions. Single histopathologic sections were also taken from the liver, kidney, and brain. The liver section showed mild to moderate sinusoidal dilation and congestion but no necrosis or granulomas. No histopathological abnormalities were noted in the kidney and brain; the spinal cord was not examined. Toxicology of femoral blood was only positive for tetrahydrocannabinol (THC). The cause of death was determined to be sudden cardiac dysrhythmia due to giant cell myocarditis with a secondary post-mortem discovery of probable pulmonary sarcoidosis.This case contains characteristics typical of GCM but raises interesting questions concerning the defining attributes of GCM, its distinction and possible relationship to sarcoidosis, and overall relation to autoimmune disease. Search criteria on PubMed included “giant cell myocarditis” AND sarcoidosis and yielded 40 results. 34 papers were omitted by the authors, resulting in six included papers describing GCM in the setting of sarcoidosis-,-,Giant cell myocarditis is a rare pathologic entity that has been reported to occur in 0.007-067% of populations across the globe.Davies et al.’sTo further grasp GCM’s complex pathology, it must be differentiated from the similar-appearing cardiac sarcoidosis (CS), which depends solely on the histopathology of the myocardium.Additionally, Okura et al.’s,,,,GCM has diverse presenting symptoms seen throughout the literature including dyspnea, fatigue, palpitations, chest pain, hypotension, headache, runny nose, and myalgias among others.-,,Clinically, GCM and CS usually distinguish themselves quite clearly. Although congestive heart failure, ventricular arrhythmias, and heart block have been associated with both CS and GCM,Cooper et al.’s,-,,,,,-Throughout the literature, GCM has been commonly associated with or suggested to be an autoimmune disorder.,,Perhaps the most influential piece of evidence supporting an autoimmune etiology of GCM was an animal study in 1990.The decedent in this case presented with psoriasis and had the post-mortem diagnosis of probable pulmonary sarcoidosis, two diseases commonly considered to be autoimmune, further supporting the relationship between GCM and autoimmunity.Giant cell myocarditis involves a mixed inflammatory infiltrate of the heart with prominent giant cells and myocyte necrosis. The disease manifests itself most often as a ventricular dysrhythmia, congestive heart failure, or sudden cardiac death but presents with a wide variety of other symptoms. The etiology of the disease has not been officially established but many have noted GCM’s autoimmune nature, so this diagnosis must be considered in patients with pre-existing autoimmune diseases, including sarcoidosis, as seen in this case. Additionally, the presence of sarcoidosis in other organs does not preclude a diagnosis of GCM.The decedent’s cause of death was giant cell myocarditis manifesting as sudden cardiac death. His symptoms were atypical, presenting only with nausea and vomiting the day prior to his death. The history of psoriasis and post-mortem diagnosis of probable pulmonary sarcoidosis revealed an association with autoimmune disorders, a characteristic seen in about 20% of reported cases of GCM. This case demonstrates a unique and rapid manifestation of GCM and raises interesting questions concerning the future research, treatment course, and complex etiology of the disease.


The reference workload for each drill was calculated across all sessions, using bootstrapping. The bootstrap mean workload and 95% confidence intervals (CI) were then calculated for session 1, sessions 1–2, and continued for remaining sessions , and were compared with those of the reference workload. The minimum sessions to anticipate workload for each drill was identified when the first normative value fell within ±5% or ±10% of the reference workload 95% CI. The minimum sessions were then tested to determine the accuracy to which workload could be anticipated. Three to four sessions were needed to anticipate workload within ±5%, while 2–3 sessions were needed to anticipate workload within ±10%. External workload was anticipated in 0–55% of future sessions using an error range of ±5%, and in 58–89% of sessions using an error range of ±10%. External workload during 5 vs. 5 games-based drills can be anticipated in most sessions using normative values established during a short-term monitoring period with an error range of ±10%.This study determined whether external workload could be anticipated during 5 vs. 5 games-based drills in basketball. Thirteen semi-professional, male basketball players were monitored during 5 vs. 5 training drills across the season. External workload was determined using PlayerLoad™ (AU∙min Basketball is a highly intermittent team sport, with players performing repeated bouts of high-intensity activity interspersed with periods of low- to moderate-intensity activity ,2. AlthoMonitoring players during both training and games is preferred to understand the complete external workloads encountered. However, monitoring external workload is more common in training than games possibly due to player preferences and restrictions in some leagues prohibiting use of wearable technologies during games . It is aGames-based drills are an effective training strategy frequently used by basketball coaches ,9. In faMicrosensor monitoring systems encompassing tri-axial accelerometers, gyroscopes, magnetometers, or a combination of these instruments , are comThirteen players  from the same semi-professional, male basketball team volunteered to participate in this study. The team was registered in the Queensland Basketball League, which is a second-tier, Australian basketball competition. Prior to study commencement, all participants were screened for injuries or health conditions that prevented safe participation in the study. The purpose of the study as well as associated risks and benefits of participation were explained to participants before providing personal and guardian written informed consent (if under 18 years). All procedures were approved by the Central Queensland University Human Research Ethics Committee (reference number: 2019-001).An observational study design was utilized whereby players were monitored during all training sessions across the 16-week, in-season phase (May–August) of the 2019 competitive season. During the season, up to 3 training sessions (2 ± 1 training sessions) were held per week (Monday–Sunday) with games played between Friday–Sunday. A total of 26 on-court, team training sessions were held across the season.Prior to commencement of the first training session, anthropometric data were collected on each player including stature using a portable stadiometer  and body mass using electronic scales . For all training sessions, players were fitted with microsensors  held on the posterior, upper surface of the torso in specially-designed neoprene shirts. All players were familiar with and accustomed to the equipment used in the present study.During each training session and following a standardized warm-up, games-based drills were conducted as part of the normal training regime determined by the coaching staff. Three distinct 5 vs. 5 drills were frequently performed by the team and thus were selected for investigation, including: (1) 5 vs. 5 played on a half-court, (2) 5 vs. 5 played on a full-court, and (3) 5 vs. 5 vs. 5 played on a full-court. Each of these drills were performed toward the end of each training session at similar time-points  following standardized shooting drills and initial games-based drills with less players . The games-based drills investigated along with associated rules and conditions are presented in x), coronal (y), and sagittal (z) planes multiplied by a scaling factor of 0.01 [−1) to account for the varied durations of each drill across the season . External workload was determined via PlayerLoad™ (PL) , which is a proprietary measure derived from the tri-axial accelerometer sampling at 100 Hz. PL represents the accumulated load calculated as the square root of the sum of the squared rate of change in acceleration across the transverse  external workload for each drill. Participation in drills were not consistent for each player across all training sessions. Further, the sample size of the study was not large. Hence we performed nonparametric bootstrapping (1000 replications) to better estimate mean workload values and a bias-corrected 95% confidence interval (CI) for each mean workload value ,23. A boThe second step involved testing the accuracy of the anticipated external workload in each drill across subsequent training sessions. We compared the 95% CI of each mean workload identified in the first step of our analyses for each drill to the 95% CI of the bootstrap mean workload experienced in each subsequent individual session. In this regard, if the 95% CI for workload in each drill in a specific session was within the acceptable error range  of the 95% CI of the workload corresponding to the minimum number of sessions identified, workload for that session was adjudged to be accurately anticipated. All statistical analyses were conducted using Stata/MP 16.0 for Windows .−1, 95% CI = 5.28, 5.85. The minimum number of sessions needed to anticipate the workload per session within ±5% for 5 vs. 5 half-court games-based drills was 3 , while the minimum number of sessions needed to anticipate workload within ±10% was 2 . Using the acceptable error range of ±5%, mean workload from sessions 1–3 did not accurately anticipate the experienced workload in any (0%) remaining sessions. Using the acceptable error range of ±10%, mean workload from sessions 1–2 accurately anticipated the experienced workload in 58% of remaining sessions , while the minimum number of sessions needed to anticipate workload within ±10% was 3 . Using the acceptable error range of ±5%, mean workload from sessions 1–4 accurately anticipated the experienced workload in 27% of remaining sessions. Using the acceptable error range of ±10%, mean workload from sessions 1–3 accurately anticipated the experienced workload in 67% of remaining sessions . Using the acceptable error range of ±5%, mean workload from sessions 1–3 accurately anticipated the experienced workload in 55% of remaining sessions. Using the acceptable error range of ±10%, mean workload from sessions 1–3 accurately anticipated the experienced workload in 89% of remaining sessions  between prescribed and experienced heart rate-derived workloads in elite field hockey players over a 5-week mesocycle. Meanwhile, Lacome, Simpson, Broad, and Buchheit [The ability to anticipate workload has only been investigated using internal workload measures and in team sports other than basketball. In this regard, Perrotta, Taunton, Koehle, White, and Warburton  reportedBuchheit  reportedBuchheit . Consequ−1) to account for the varied durations of each drill across sessions. In this sense, variation in drill duration likely influenced the external workload intensities experienced by players, with basketball players shown to accomplish higher training intensities when external workload is sampled across shorter durations [We showed normative external workloads could be established using only 3–4 training sessions when using an acceptable error range of ±5% of the 95% CI for the mean workload obtained across the entire season. However, our data demonstrate relatively poor accuracy (0–55%) when using an acceptable error range of ±5% to anticipate external workload during 5 vs. 5 games-based drills. The session-to-session variation in external workload using this error range was likely due to the workload management strategies implemented by coaching staff  , as wellurations . ConsequIn contrast, we showed normative external workloads could be established using only 2–3 training sessions when using an acceptable error range of ±10% of the 95% CI for the mean workload obtained across the entire season. Consequently, the more liberal acceptable error range of ±10% yielded greater accuracy (58–89%) in anticipating the external workload experienced during 5 vs. 5 games-based drills. Accuracy may however be dependent upon the drill used as the number of sessions accurately anticipated was just over half (58%) for 5 vs. 5 half-court drills, which increased to 67% and 89% for 5 vs. 5 and 5 vs. 5 vs. 5 full-court drills, respectively. The greater accuracy in anticipating external workload in full-court games-based drills might be due to completion of more consistent running demands during transitions across the court than half-court games-based drills, which evoke more play sequences close to the basket underpinned by variable movement patterns  and tech−1; full-court: 17.9 ± 4.6 AU·min−1) and Montgomery et al. [−1) reported higher relative external workloads than we measured during 5 vs. 5 games-based drills in professional, adult and elite, junior, male players, respectively. However, variations in accelerometer placement across studies likely accounts for these differences given past studies placed the accelerometers on the hips [−1 and 1.0 ± 0.1 AU·min−1 during half- and full-court 5 vs. 5 games-based drills in professional, male players. Despite these data being approximately tenfold lower than the external workloads we reported, differences in the monitoring system, algorithm, and sampling rate used by Vazquez-Guerrero et al. [The present data add to the limited body of knowledge surrounding the external workloads experienced during various 5 vs. 5 games-based drills using microsensors. The mean external workloads we observed vary from results reported in previous studies. Specifically, Schelling and Torres   and RPE  than continuous games-based drills. Consequently, the inclusion of short break periods permits coaches to reduce the demands imposed on players if desired by lowering the work:rest ratio encountered [Our study also quantified the external workload experienced during a novel 5 vs. 5 vs. 5 games-based drill. This drill is practically advantageous for basketball coaching staff as it allows implementation of short break periods, which resemble the intermittent nature of basketball game-play. Furthermore, as previously shown by Conte, Favero, Niederhausen, Capranica, and Tessitore , intermiountered .Although our study provides important information regarding the use of microsensors to quantify and anticipate external workload during 5 vs. 5 games-based drills in basketball, some limitations should be considered. (1) The occurrence of the included 5 vs. 5 games-based drills could not be controlled, resulting in varied sample sizes, in addition to a relatively small sample size, for analysis per drill. Nevertheless, the varied sample sizes is a consequence of conducting applied studies in actual training scenarios, which strengthens the ecological validity of our outcomes. Further, we used bootstrapping, a resampling technique, in an attempt to better estimate the parameters of workload. (2) Where drills were performed more than once in a session, only the first occurrence of the drill was utilized for analysis to control for the confounding influence of fatigue on workloads. Consequently, the inability to anticipate the external workload we observed only applies to the first occurrence of each drill in each session. (3) Drill length was not controlled for across sessions. Future research is encouraged to investigate the potential relationship between drill length and intensity during different games-based drills in basketball. (4) Given the observational nature of the present study, the timing of the included 5 vs. 5 games-based drills within each session could not be controlled. Consequently, the 5 vs. 5 games-based drills were administered ~40–56 min into each session on average. Further research is encouraged to determine whether external workload can be more accurately anticipated during games-based drills when administered at other timepoints during training sessions. (5) Player activity prior to and following each training session was not controlled. However, team training schedules remained consistent across the season, likely allowing for similar recovery and preparation leading into each session. Furthermore, we assessed the ability to anticipate external workload during games-based drills across the season in a natural team training environment to enhance the translation of findings to practical settings. (6) Acceptable error ranges of ±5% and ±10% were included in this exploratory analysis; however, some coaching staff may be more conservative or liberal in their approach, which warrants further research using alternative acceptable error ranges.Normative values obtained across 3–4 sessions showed limited accuracy (0–55%) in anticipating external workload using an acceptable error range of ±5% during 5 vs. 5 games-based drills in semi-professional, male basketball players. However, normative values obtained across 2–3 sessions accurately anticipated external workload across most sessions (58–89%) using an acceptable error range of ±10% during the same drills. Consequently, external workload can be anticipated in most instances during 5 vs. 5 games-based drills using normative values established during a short-term monitoring period using an acceptable error range of ±10%.Our findings have important implications for basketball coaching staff when implementing microsensor monitoring systems in practice. The ability to anticipate external workload using a small sample of sessions to establish normative values allows for the short-term uptake of microsensor monitoring systems, reducing the extensive financial cost, labor, and expertise associated with the long-term monitoring of players across the entire season. In addition, the normative external workloads we provided for different 5 vs. 5 games-based drills in basketball should be considered by coaches when developing training sessions across the season. The novel 5 vs. 5 vs. 5 drill examined in this study may provide a useful training stimulus for coaches when seeking to incorporate short rest periods between active play and up to 15 players in a large team environment, which is important given bench players can complete less training demands than starting players in basketball settings .


Staphylococcus aureus (S. aureus) is an opportunistic pathogen capable of causing serious health implications in susceptible individuals once it invades the host’s protective barriers. Methicillin-susceptible S. aureus (MSSA) often receives lesser attention although it has been frequently associated with serious infections in human. We aim to investigate the genomic features of a highly virulent yet pan susceptible MSSA strain (coded as HS-MSSA) which caused concurrent bacteraemia in a dengue patient, ultimately resulted in sepsis death of the patient. Whole genome sequence analysis was performed. The draft genome of HS-MSSA is approximately 2.78 Mb (GC content = 32.7%) comprising of 2637 predicted coding sequences. In silico genotyping of the HS-MSSA strain revealed a novel combined genotype (t091/ST2990). The HS-MSSA carries a SaPIn1-like pathogenicity island that harbours the staphylococcal enterotoxin and enterotoxin-like genes (sec3 and selL). The strain-specific β-lactamase (blaZ)-bearing plasmid region was identified in HS-MSSA. Core genome phylogeny showed that the HS-MSSA strain shared a common ancestry with the European MRSA clone. We report herein the genomic features of an MSSA lineage with novel genotype previously not reported elsewhere. Staphylococcus aureus (S. aureus) is a Gram-positive round-shaped bacterium commonly found colonizing the skin and mucous membranes of humans. Approximately 15% of the human population carry S. aureus in the nasal cavity, specifically in the anterior nares1. S. aureus is an opportunistic pathogen capable of causing serious health implications in susceptible hosts when it enters the bloodstream or internal tissues. Potentially serious infections caused by S. aureus include bacteraemia, pneumonia, endocarditis, and osteomyelitis2. In fact, the nasal carriage of S. aureus has shown a strong association with the aetiology of bacteraemia in susceptible hosts3. Bacteraemia caused by invasive S. aureus often results in greater mortality rate due to its ability to secrete a wide spectrum of virulence factors that contribute to the evasion of host immune responses4.S. aureus infections are often associated with healthcare facilities due to the breaching of the skin barrier with surgical devices or implants, although it is not uncommon to have community acquired infections5. Antimicrobial resistance (AMR) among the healthcare-associated S. aureus infections has been an important global public health issue. Since the emergence of methicillin-resistant S. aureus (MRSA) in the 1960s, this organism has been notorious for its ability to cause severe forms of infection in hospital settings and has eventually spread to the community after 30 years of evolution, most probably due to the transfer of the AMR gene cassette SCCmec to local methicillin-susceptible lineages6. This evolutionary theory for the emergence of MRSA lineage has recently been strengthened by whole genome sequence analysis7.The severe consequences of S. aureus (MSSA) infection has persisted in both healthcare settings and the community, amidst the increasing global prevalence of MRSA8. In fact, a slow epidemic shift was observed in S. aureus infections worldwide during the past two decades. In the United States (US), multiple clinical studies have recorded an increase in the prevalence of MSSA and a simultaneous decrease in MRSA incidences since 200510. Recent surveillance in the US has seen an increase in bloodstream infections caused by community acquired MSSA from 2012 to 201711. A similar observation was recorded in Europe, whereby a decrease in MRSA bacteraemia was observed since 2005, accompanied by a continuous increase in MSSA bacteraemia12.Despite receiving less attention compared to its drug-resistant counterpart, the methicillin-susceptible 15. More often than not, invasive MSSA causes greater illness severity in patients suffering from bacteraemia due to its ability to express multiple virulence factors17. Community-onset MSSA bacteraemia has documented a higher mortality rate compared to that of MRSA in many regions18. These observations showed that while much attention has been directed to MRSA infections in recent years, the often neglected MSSA infections have caused a greater and increasing disease burden worldwide.Genotyping studies from different geographical regions often revealed a greater genetic diversity among the MSSA populations compared to the MRSAThe ability of the diverse MSSA lineages to express a multitude of virulence genes and the possibility of developing multi-drug resistance phenotypes due to horizontal gene transfer have posed a great risk to public health. Nonetheless, the genetic drives that are responsible for the emergence and persistence of such virulent MSSA lineages remain obscured. In this study, we aimed to investigate the genomic features of an invasive MSSA strain which caused a fatal sepsis case in human. The patient was initially diagnosed with dengue fever upon admission to the hospital, but later developed pneumonia and severe sepsis rapidly and succumbed to multiple organs failure within 24 h post-admission.19.The community acquired MSSA strain (coded as HS-MSSA) was isolated from the blood culture of a patient in 2017. The clinical presentations as well as the AMR and virulence characteristics related to the infection have been previously described20. The HS-MSSA genomic DNA was subsequently subjected to next-generation sequencing via Illumina Genome AnalyzerIIx  by a commercial sequencing vendor. Paired-end 2 × 150 bp reads were generated using Illumina chemistry. The quality of the sequence reads was assessed and assembled de novo using the CLC Genomics Workbench version 5.1  with default parameters. Contigs with an average coverage of 30 and above were selected to represent the bacterial draft genome for further analyses. Reordering of the HS-MSSA contigs was achieved via genomic mapping with the reference genome S. aureus subspecies aureus NCTC 8325  GenBank accession number: NC007795) by using Mauve software version 2.4.021. The subset of the contigs that were mapped to the complete chromosome of the S. aureus NCTC 8325 was identified as the chromosomal region of the HS-MSSA strain. Genomic annotation was performed using the webserver Rapid Annotations using Subsystems Technology 22. The viewing and preliminary sequence-based comparison between the annotated HS-MSSA and reference genomes were done using SEED Viewer version 2.0 (http://pubseed.theseed.org/)23, and further validated by using Mauve.The genomic DNA of the HS-MSSA strain was extracted using DNeasy Blood & Tissue Kit  and the quality of the extracted genomic DNA was evaluated based on spectrophotometric measurementshttps://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify genetically similar S. aureus strains (taxonomy ID: 46170) from the complete and draft genomes database curated in the NCBI GenBank. The BLAST search was performed using the discontinuous megablast algorithm with default parameters. Multiple genomes alignment and visualization were performed using Mauve and BLAST Ring Image Generator (BRIG) software24. The genotype of the HS-MSSA strain was inferred in-silico by using web-based servers. The conventional seven-gene multi-locus sequence typing (MLST), whole genome MLST (wgMLST), and ribosomal MLST (rMLST) predictions made use of the S. aureus database in PubMLST (https://pubmlst.org/saureus/) sited at the University of Oxford25. The online analysis tools available from the Center for Genomic Epidemiology (CGE) server (http://www.genomicepidemiology.org/) were used to predict the staphylococcal protein A (spa) type, presence of SCCmec elements, plasmid elements, and pathogenicity genes in the HS-MSSA genome29. Phage-associated genes and genomic regions in the HS-MSSA genome was identified using PHAge Search Tool Enhanced Release server 31. The genomic islands were predicted using IslandViewer 4 (http://www.pathogenomics.sfu.ca/islandviewer/)32. All genomic predictions were completed using default parameters, validated by cross-examination with RAST-annotation output, and manually interrogated using NCBI BLAST nucleotide search tool.The chromosomal region of the HS-MSSA was subjected to a microbial genome search using the NCBI Basic Local Alignment Search Tool  was used to identify AMR genes in the assembled HS-MSSA genome33. The AMR genes selection criteria were set to perfect (100% identity) and strict (> 95% identity) hits only to the curated reference sequences in the Comprehensive Antibiotic Resistance Database . Prediction of partial genes was accepted as the HS-MSSA genome examined in this study resembles an incomplete genome. The RGI predictions were cross-checked with ResFinder version 3.2 (https://cge.cbs.dtu.dk/services/ResFinder/). AMR genes prediction results obtained using default parameters34. All predicted AMR genes were validated by cross-examination with RAST-annotation output and manually interrogated using NCBI BLAST.The online tool, Resistance Genes Identifier version 5.1.0  was used to identify relevant single nucleotide polymorphism (SNP) sites for core genome phylogenetic analysis35. Complete and draft genomes of 20 global S. aureus strains were selected for core genome SNP-based (cgSNP) phylogenetic analysis based on considerations including high genetic identity , geographical distribution, type of organisms (MSSA and MRSA), and MLST clonal complexes. The selected S. aureus genomes were retrieved from the NCBI GenBank and PubMLST databases and the strains information is tabulated and appended as Supplementary Table S. aureus NCTC 8325 was used as the reference genome for multiple alignments. The multiple genomes sequence alignment generated by the REALPHY tool was subsequently used to construct a phylogenetic tree. An unrooted tree was inferred via the approximate maximum likelihood (ML) method using FastTree2 software36, and visualized using FigTree version 1.4.3 (https://github.com/rambaut/figtree/releases)37. 100 bootstrap replicates were performed to support the phylogenetic inferences made in this study for practical reasons. A bootstrap analysis with 100–500 replicates is sufficient to produce qualitatively comparable support values with more than 99.5% correlation with the reference values in ML trees with higher numbers of bootstrap replicates38.The online tool, Reference sequence Alignment based Phylogeny builder version 1.12  for the central STs of the major clonal complexes  and other frequently encountered clonal complexes  were used to construct a phylogenetic tree. Multiple alignments of the concatenated sequences were performed by Molecular Evolutionary Genetics Analysis (MEGA) X39. Next, the aligned sequences were subjected to a statistical selection of best-fit phylogenetic model using jModelTest version 2.1.640. An unrooted tree was subsequently inferred via the ML method using MEGA X software.The concatenated allelic sequences of the seven housekeeping genes  and 65 RNAs were predicted in the HS-MSSA draft genome. The general features of the HS-MSSA draft genome are summarized in Table The size of the HS-MSSA draft genome is approximately 2.78 Mb (GC content = 32.7%), of which 2,740,810 bp belongs to the genomic region that corresponds to the chromosome of S. aureus chromosomal regions. Microbial genome BLAST analysis showed that the HS-MSSA chromosome shares greatest genetic homology  with the MRSA strains isolated in the United States of America (USA). More than half of the MRSA strains that are genetically similar to the HS-MSSA were obtained from a clonal cluster of community acquired MRSA that caused skin and soft tissue infections (SSTIs) in a military base in the USA41. Similar genetic homology was observed between the MRSA USA300 clones and HS-MSSA, albeit at slightly lower scores. The two USA300 clones that showed greatest genetic homology with HS-MSSA are USA300_TCH1516 (NC_010079) and USA300_SUR1 (NZ_CP009423), both being community acquired MRSA strains isolated from USA and Republic of Suriname in South America respectively. A circular genome map was constructed to illustrate the genetic resemblance of HS-MSSA to the representative MRSA clones from the North and South America (MRSA strains 2148.C01 (NZ_CP017094) and USA300_SUR1), and an MSSA strain KT/314250 (NZ_AOCP00000000) isolated in Malaysia  that caused an outbreak of SSTIs shared common MLST type (ST8) with the community acquired USA300 clone from Texas, USA43. The complete genome analysis revealed that the South American outbreak strains were genetic variants of the North American USA300 clone45. Previous genomic studies have proven that USA300 clones are clonally related to the European nosocomial MRSA strain COL (NC_002951) emerged from a methicillin-susceptible ancestral strain via the acquisition of methicillin resistance determinant following the introduction of methicillin in 195946. Therefore, the MRSA clones USA300 and COL are most likely sharing a common MSSA ancestry but evolved separately through different MRSA lineages47. Accordingly, the close genomic resemblance of the HS-MSSA strain to these MRSA clones suggests that this MSSA lineage in Malaysia could have been the less evolved (with reference to AMR) descendant of the common MSSA ancestry with the MRSA clones in the USA and Europe. This inference was made on the basis that chromosomal recombination occurs in S. aureus at low frequencies, except for regions associated with virulence and pathogenicity48. The descendants of this MSSA ancestral strain have spread widely over different continents and persisted in the human populations over the years, causing community acquired SSTIs and invasive infections in the susceptible hosts.The great genomic resemblance between the HS-MSSA strain and the MRSA clones from the American continent indicates the possible common ancestry between these lineages, despite the wide geographical distance where the strains were originated. The MRSA clones originated from the USA that showed high genetic homology with HS-MSSA were all community acquired, capable of causing SSTIs and sepsis, and were largely resistant to chlorhexidinespa type t304 and MLST type ST6 (clone t304/ST6) which caused recurrent outbreaks in a hospital in Denmark since 201149. This MRSA clone t304/ST6 was detected and predominated among the MRSA strains isolated in a teaching hospital in Oman within the same period50. However, this clone remains rarely reported outside these two geographical regions. The finding of similar plasmids in the HS-MSSA strain in Malaysia and MRSA clone in European region suggests possible horizontal gene transfer event, which could have occurred due to chance events after the divergence from the common MSSA ancestral strain. One other hypothesis is that the HS-MSSA strain could have evolved from an MSSA lineage carrying this plasmid, of which some of its descendants might have diverged to another evolutionary path via the acquisition of SCCmec gene cassette. The latter hypothesis is more plausible as this plasmid carries the penicillin-resistant blaZ gene. Based on an earlier study using Staphylococcus species mainly from the bovine origin, the authors demonstrated that plasmid-borne blaZ is often species-specific and lack conjugative transfer mechanisms, thus making horizontal gene transfer an extremely rare event51. Therefore, the presence of this blaZ-bearing plasmid in the HS-MSSA strain supports our inference on the common ancestry of this local MSSA with the MRSA clones in European regions.Detailed examination of the 15 contigs that were not included in the HS-MSSA chromosomal region revealed a 19,763 bp region (contig-33) identified as a plasmid-associated genomic region. BLAST analysis of contig-33 revealed the closest plasmid homolog as pl1_M2024 (CP047022), at 100% query coverage with 99% nucleotide identity . The pl1_M2024 is a complete plasmid retrieved from the nosocomial MRSA clone with S. aureus by rMLST. The 7-loci allelic profile for HS-MSSA is 1-1-1-1-330-1-10, arranged in the order arcC-aroE-glpF-gmk-pta-tpi-yqiL. All loci MLST (wgMLST) results showed 1358 exact matches to sequence type ST2990 (Supplementary Table S. aureus strain 128A deposited in the database (PubMLST ID: 5521) shares the same sequence type as HS-MSSA and was also isolated in Malaysia, earlier in 2014. S. aureus 128A was isolated from the infected wound of an elderly diabetic patient (66 years old) examined in a tertiary hospital in the capital Kuala Lumpur. The strain was found susceptible to oxacillin, methicillin, and vancomycin upon investigation. The S. aureus 128A was confirmed as community acquired MSSA which causes wound infection. The spa type of HS-MSSA is t091 (spa repeat succession 07-23-21-17-34-12-23-02-12-23). Therefore, the combined genotype of the HS-MSSA strain is noted as t091/ST2990 (spa type/MLST type).In silico MLST analyses showed that HS-MSSA belongs to the sequence type ST2990, with its identity confirmed as S. aureus strain 128A which shares the identical sequence type (ST2990), the HS-MSSA strain examined in this study was isolated from a diabetic patient who was admitted to the hospital due to dengue fever19. However, instead of causing SSTI (infected diabetic wound) as strain 128A did, the HS-MSSA was more invasive and caused a bloodstream infection that resulted in severe sepsis. The common STs of S. aureus in Malaysia, especially the most studied MRSA strains, mainly comprised of the pandemic clone ST239, with ST22 clone being increasingly documented in the past decade54. Similar to the global epidemiology of MSSA, the few studies that examined the MSSA genotypes in Malaysia documented a higher genetic diversity compared to the MRSA populations in this region57. MRSA with spa type t091 has been reported in Malaysia, but S. aureus strain with genotype t091/ST2990 has not been previously documented58. Nonetheless, the isolation of ST2990 strains from different geographical regions suggests that this MSSA genotype may already be widely disseminated in Malaysia. Although S. aureus of CC1 complex  does not predominate in Malaysia, the persistence of these genotypes throughout the years may indicate their evolutionary success58.Similar to the mec (SCCmec) elements were not detected in the HS-MSSA genome based on SCCmecFinder analysis at 90% identity threshold and a minimum length of 60%. The absence of SCCmec elements in the HS-MSSA genome is reflected in the methicillin-susceptible phenotype of this strain. The SCCmec genomic cassette is a mobile genomic region with two important gene complexes, mec-gene complex encoding methicillin resistance and cassette chromosome recombinase (ccr)-gene complex encoding site-specific recombinase(s) for the mobility of this DNA region59. The presence of SCCmec region in the bacterial chromosome is a signature of MRSA strains and the acquisition of which results in the emergence of new MRSA lineage from an ancestral MSSA strain.The staphylococcal chromosome cassette mec elements provides a selective advantage to S. aureus, it is not uncommon for it to spontaneously excised from the MRSA in human hosts60. Partial excision is sufficient to result in methicillin-susceptible phenotype, and most often such MSSA strains showed great genetic homology to its MRSA counterpart, although less prevalent61. Given the genomic resemblance of the HS-MSSA strain to the global MRSA clones, we decided to investigate whether such an excision event has occurred in this local MSSA strain. In order to identify possible remnants of SCCmec cassette or its homologs in the genome, the database search was repeated at 60% identity threshold with 30% minimum query cover length. The search result remains the same, i.e. intact or partial mec genes and the whole cassette was not detected, but a 1.5 kb DNA region associated with the insertion sequence-like IS1272 in SCCmec type I was detected in contig-4, albeit with considerable genetic variation (94% query coverage and 82% nucleotide identity). BLAST analysis of this 1.5 kb region revealed the presence of a pseudogene of IS1182 family transposase in S. aureus . In short, none of the SCCmec elements was detected except for the 1.5 kb DNA region homologous to IS1272 commonly found in the mec-gene complex in MRSA that may also present infrequently in MSSA62.While the acquisition of SCC19. Genomic sequence analysis revealed a low number of AMR-conferring genes compared to virulence genes, and a majority of which are involved in bacterial efflux systems . Cross-examination with virulence genes analysis showed that the HS-MSSA strain carries multiple genes encoding for bacterial adherence, enzyme production, host immune evasion, various prominent staphylococcal toxins, a type VII secretion system, and intact regulatory operons for virulence expression . The virulence analysis of the HS-MSSA strain is reported in detail in our earlier publication19  in the MRSA represses the transcription of the agr operon, thus halting the production of the virulence factors that are regulated by this operon. Therefore, the ability of the HS-MSSA to cause severe sepsis in the human host could be related to the lack of SCCmec genes cassette in its genome, thus the expression of its multiple virulence factors was not impeded.The pathogenicity genes analysis showed that the HS-MSSA harbours 536 CDSs which matched to known lukFS-PV). When mapped to the reference genome S. aureus NCTC 8325, the PR2 showed great genetic similarity with a 72 kb prophage on NCTC 8325. The PR3 was identified on contig-11, closely associated with genes encoding for β-hemolysin (hlb), cytolytic pore-forming protein (lukGH), extracellular adherence protein (eap), and staphylococcal complement inhibitor (scn). A total of six genomic islands (GI1-GI6) were identified in the HS-MSSA chromosome by using S. aureus NCTC 8325 as the reference 67. An array of staphylococcal virulence genes mainly associated with bacterial adherence and stress response were found in GI4, while GI5 consisted of mainly hypothetical proteins.Three prophage regions were identified in the HS-MSSA chromosome. Phage region 1 and 3 (PR1 and PR3) are incomplete prophages, while phage region 2 (PR2) is an intact prophage spanning a 64.1 kb genomic region , sharing of similar mobile genomic regions , same geographical origin (KT/314250), same clonal complex (MSSA476), and representative MSSA and MRSA genomes (MSSA476 and COL) from European region . The HS-MSSA genome showed ≥ 94% genome coverage and ≥ 99.16% nucleotide identity  with all selected S. aureus genomes. The ST22 reference genome HO 5096 0412 (NC_017763) from a clonal complex (CC22) less closely related to other S. aureus strains was used as an outgroup in constructing the ML tree.Representative 67. Together with another MRSA strain M2024 (NZ_CP047021) which caused recurrent clonal outbreaks in Denmark49, the three S. aureus strains formed a loose cluster with relatively divergent evolutionary paths and geographical origins (Cluster I). Interestingly, the MSSA476 from the United Kingdom that shares a common clonal complex (CC1) with HS-MSSA formed another distinctive cluster (II) although both are community acquired MSSA capable of causing bacteraemia in human hosts and carried the strain-specific plasmid-borne blaZ gene70. Similarly, the MSSA strain KT/314250 which was also isolated in Malaysia formed a separate clade distinctive from the HS-MSSA lineage but is tightly clustered with the MSSA476 strain. The representative S. aureus strains from Europe (NCTC 8325 and COL) and MRSA clones from America formed a third cluster (III) more distantly related to the HS-MSSA strain.The phylogenetic analysis results revealed that the HS-MSSA strain formed a distinctive clade by itself, sharing a more recent common ancestry with the MRSA clones in Europe and Japan (M2024 and N315)  with HS-MSSA only appeared on scientific records in 2014. Based on the limited strain data available in the PubMLST database for the MSSA strain 128A, we hypothesize that the ST2990 strains in Malaysia showed variable clinical manifestations, ranging from non-invasive SSTI to severe bacteraemia. The HS-MSSA lineage has probably accumulated sufficient adaptive features through the acquisition of mobile genetic elements, giving rise to its unusual virulence in causing severe invasive infection in human host19. Moreover, the close genetic proximity with the MRSA clones and its genome plasticity also suggests that the HS-MSSA lineage could provide a stable genetic environment to allow the acquisition of genetic elements that confer drug resistance6. Therefore, continuous molecular surveillance to detect highly virulent MSSA in clinical settings is essential to allow timely management of invasive staphylococcal infections and the development of potential anti-virulence therapy.The phylogenetic relationships of the HS-MSSA strain with the global S. aureus strains selected for cgSNP-based phylogenetic analysis, we observed that the HS-MSSA strain genotyped as CC1/ST2990 was not found on the same clade as other CC1 strains. In order to investigate the accuracy of this phylogenetic clustering, representative genomes of the central STs for the major CCs were retrieved from the PubMLST database for further analysis71. Overall, the clustering of the STs corresponded with the CC grouping based on the 7-loci MLST scheme  which involves the assessment of 1351 loci (for S. aureus) and the whole genome MLST (wgMLST) are the more appropriate approaches for inferring phylogenetic relationship of genetically clonal strains. Previous studies using different bacterial species have observed a high concordance between cgMLST and wgMLST with SNP-based analysis, generating phylogenetic trees with great statistical similarity and almost equivalent clustering of bacterial strains73. Therefore, the current clustering of ST2990 into CC1 needs to be re-assessed by the scientific community. Nonetheless, more ST2990 genomes should be included for cgSNP-based analysis in the future to verify this notion. The present study is limited by only one ST2990 (HS-MSSA) genome that was available on publicly accessible genomic databases at the time of writing.Our observations support the previous notion that the current grouping of We report herein a novel genotype of MSSA (t091/ST2990) of community origin that caused severe sepsis in the human host. Upon examination, the HS-MSSA genome harbours only very few AMR genes, which was reflected in its pan-susceptible phenotype. The HS-MSSA strain carries multiple virulence genes in its genome, including prominent staphylococcal toxin genes that are associated with mobile genetic elements (prophages and SaPI). The HS-MSSA strain shares a common ancestry with the MRSA clone originated from the European region, but instead of developing drug resistance, it has evolved through a different path via the acquisition of multiple virulence and pathogenicity factors. Due to the limited epidemiological data available for this novel genotype (t091/ST2990), we could not conclude that this MSSA lineage has emerged locally or the HS-MSSA strain was just a sporadic strain imported from other regions without molecular typing data. Although the patient reported no history of recent travels upon admission, we cannot rule out the possibility of local transmission. Therefore, increased effort in molecular surveillance is essential to allow the development of appropriate treatment strategies to reduce the mortality risk of severe invasive infections caused by this organism.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


Graphene has become the focus of extensive research efforts and it can now be produced in wafer-scale. For the development of next generation graphene-based electronic components, electrical characterization of graphene is imperative and requires the measurement of work function, sheet resistance, carrier concentration and mobility in both macro-, micro- and nano-scale. Moreover, commercial applications of graphene require fast and large-area mapping of electrical properties, rather than obtaining a single point value, which should be ideally achieved by a contactless measurement technique. We demonstrate a comprehensive methodology for measurements of the electrical properties of graphene that ranges from nano- to macro- scales, while balancing the acquisition time and maintaining the robust quality control and reproducibility between contact and contactless methods. The electrical characterisation is achieved by using a combination of techniques, including magneto-transport in the van der Pauw geometry, THz time-domain spectroscopy mapping and calibrated Kelvin probe force microscopy. The results exhibit excellent agreement between the different techniques. Moreover, we highlight the need for standardized electrical measurements in highly controlled environmental conditions and the application of appropriate weighting functions. One of the most promising methods is the use of CVD to grow graphene onto various metallic substrates. In CVD growth, graphene is grown on the surface of the metal after hydrocarbons decompose2. The most promising metal substrate used until now is Cu3, however graphene is currently grown also on Ni(111)6, Ru(0001)7, Pt(111)9, and Ir(111)11. Subsequently to the growth process, for graphene to be use in electronic applications, it needs to be transferred on an insulating substrate , which is a transparent and flexible substrate). The already happening now use of graphene in RF electronics12, integrated circuits13 and optoelectronics14 has triggered impressive progress in large-scale production, however the community still lacks standardised electrical measurements to extract useful parameters such as carrier concentration, mobility and sheet resistance, all of those are often presented as figures-of-merit of the graphene quality. Currently, the most widely used method for electrical characterisation involves magneto-transport measurements in lithographically patterned Hall bars to extract carrier concentration, mobility and resistance. However, this method is not suitable for high throughput characterisation and often the measured graphene is significantly altered due to the microfabrication processes, in which case the electrical properties of the pristine transferred graphene are different from the ones measured. An alternative method for mapping the sheet resistance of graphene films is using micro four-point probes (M4PP)16. Recently the use of microwave cavities18 and Terahertz time-domain Spectroscopy (THz-TDS)23 measurements provided a non-contact method of measuring the conductivity of graphene films. Despite the attractiveness of these techniques, fluctuations in ambient conditions (such as humidity) affect the electrical properties of graphene25, resulting in discriminations between measurements at different laboratories.The unique properties of graphene26. This method was originally introduced by van der Pauw in 1958 to measure the resistivity and Hall coefficient of arbitrary shape semiconductors28.In contrast to the conventional Hall effect, where device fabrication is required for carrier concentration and mobility measurements, the van der Pauw (vdP) method only requires thin uniform samples with relatively small Ohmic contacts31.THz-TDS has recently been demonstrated to be an excellent tool to characterize the quality and conductivity of graphene deposited on large areas with suitable choices of substrates and models. THz-TDS can provide a map of the absolute value of conductivity/resistivity in a non-contact and non-destructive manner. The measurement is fast and can cover the entire area of a sample and can be used in all stages of a material development since the prototyping up to manufacturing scale32 is commonly used for measuring the local work function and distribution of graphene layers with nanometre resolution. Nevertheless, this technique requires ultra-high vacuum to operate and the surface must be completely clean, meaning that high temperature annealing (~400 °C) is used prior the measurement. Both conditions pose a significant drawback when rapid characterisation is needed. An alternative, complementary technique that can provide valuable information about local electrical properties, such as surface potential, doping and work function as well as providing essential information about layer distribution down to ~20 nm spatial resolution is frequency-modulated Kelvin probe force microscopy (FM-KPFM)34. KPFM has already demonstrated great potential for graphene characterisation, identification of graphene layers as well as measuring the surface potential of the samples with nanometre resolution40.An important property of graphene is its work function 41. They will become part of Good Practice Guides for the measurement of electrical properties of graphene and will be proposed as an input to normative bodies, towards the development of documentary standards. In this work, we demonstrate appropriate measurement techniques to characterise the most important electrical properties of graphene from macroscale down to nanoscale and establish the validity of the measurements through correlation in an inter-laboratory study and applying a weighting correction function between the results obtained from THz-TDS and van der Pauw methods.In this paper, we propose and demonstrate a standardised procedure for measuring and mapping the electrical properties of scalable graphene using a combination of contact and contactless complementary methods, such as van der Pauw magneto-transport, THz-TDS conductivity mapping and Kelvin probe force microscopy. The methodologies here described are currently being developed within the GRACE projectIntensity-Z curves for each pixel from the stack of images. The system is operated in ambient air and does not require any sample preparation. For these measurements, individual images with high magnification (100 × objective) were captured and then stitched together to generate a high resolution image of a larger area42.Confocal laser scanning microscopy (CLSM) is a powerful optical technique that enables rapid structural characterisation of large-area graphene and graphene nanostructures by pushing the spatial resolution beyond the optical diffraction limit and stacking individual images of different focal planes. CLSM was performed using an Olympus LEXT OLS4100 system equipped with 5×, 10×, 20×, 50× and 100× objectives  and with further 1× to 8× optical zoom. Therefore, images of a field-of-view ranging from 2560 μm to 16 μm can be captured. The system uses a 405 nm wavelength laser, which scans in X-Y directions using an electromagnetic micro-electromechanical system (MEMS) scanner and a high-precision galvano-mirror. The optical images  are generated using a photomultiplier which captures the reflected light. In this confocal setup, in-focus reflected light is only allowed to pass through the circular confocal pinhole, therefore eliminating flare from out-of-focus areas. Due to the shallow depth-of-field of the CLSM setup, the objective is moved vertically to capture images of different focal planes. Using this Z-stacking technique, a single output of the 2D reflected intensity map is extracted by determining the maximum brightness value of the calculated f0 ≈ 300 kHz, with an AC voltage of much lower frequency fmod ≈ 3 kHz of 5 V amplitude also applied, to induce a frequency shift of f0 ± fmod. The side lobes (monitored by a PID feedback loop) generated by this shift are minimized by applying a DC compensation voltage. By measuring this DC voltage at each pixel, a surface potential map [i.e. contact potential difference . Since FM-KPFM measures the surface potential (difference in work function between the tip and the sample), it provides a qualitative result, highly dependent on the tip status. In our approach we demonstrate a method to appropriately calibrate the work function of the scanning tip (24). Then the same HOPG reference sample is measured using FM-KPFM, and the tip is calibrated using FM-KPFM uses the force gradient mentclass2pt{minimRsTHz) is based on measuring the difference of the THz reflectivity of the film on top of the substrate and the reflectivity of the bare substrate. In transmission geometry, the conductivity is extracted by comparing the signal of the sample with the thin film and the signal of the bare substrate in transmission mode.The extraction of the resistivity of a thin film on a substrate using THz-TDS in reflection geometry  of the sample, passing current and measuring voltages in different configurationsBy applying current and measuring the voltage drop at the opposite sides and repeating the operation by rotating the sample by 90° Fig. , 8 resisUsing the values from Eq. , the resTherefore, the sheet resistance VH) is measured between the remaining two corners  and mobility (μ) can be calculated using the following equations from the Hall coefficient RH:Finally, the carrier concentration  response of the AC biased sample is measured. In both VH measurements, the voltages are measured using a lock-in amplifier (LIA) referenced to the first harmonic of To measure the magneto-transport properties in vdP geometry, an Arduino microcontroller is used to trigger solid state relays, allowing current to pass and voltage measurements to be done at the different configurations as shown in Table ing Eqs.  and 8)..\documenρ(P) is the local sheet resistivity at point P, 47 to identify the general form of ρ(P) itself. The zero-order approximation for 47), with a value of about 3.4 in the centre and vanishing on the square edges. Therefore, the resistivity near the centre affects The sheet resistance values In the following, we present and discuss results obtained on a batch of 6 samples of CVD graphene transferred on a single quartz substrate and diced into chips.Figure 25. The summarized results of work function measurements of the different samples are shown in Fig. When a single-atomic layer such as graphene is measured, surface contamination may lead to incorrect results. When graphene is transferred on a target substrate, the top surface is covered with polymer residues. In our measurements, the residues are removed prior to the measurement using contact-mode atomic force microscopy (AFM) to expose a clean surface. The results in ambient are shown in Fig. Figure entclass1pt{minima48, and they can be related to the presence of microscopic defects, such as cracks, and to the presence of growth domain. The measurements are affected by edge effects that span about 1 mm from the edge due to the size of the THz beam, which is estimated to be 0.5 mm in diameter. Most of the features show up as spots or islands of significantly higher sheet resistance, indicating a low quality of the graphene deposited in that particular area . Other features show up as two distinct sheet resistance areas separated by a varying size transition zone (e.g. samples 3 and 39).The features observed in these maps have been reported in the literature in25. Additional advantage of the vdP method is that using the current setup both ambient and vacuum measurements can be performed and thus the effect of the environmental exposure on the electrical properties of graphene can be studied.Following the THz-TDS sheet resistance mapping, the magneto-transport properties of six samples were measured using the home-build vdP setup described in Section 2.4. VdP can be considered as a complementary method to THz-TDS measurements as the later one only provided information about the sheet resistance/conductance and can only be performed in ambient conditions. As both carrier concentration and mobility are essential parameters for the material performance, their measurements provide crucial information about the quality of the graphene layer25. Therefore, using the dedicated measurement setup, the magneto-transport measurements in the van der Pauw geometry were also performed in vacuum. Prior the measurement, the samples were annealed (170 °C) in vacuum  Fig. , such asers Fig. .Figure 6In order to compare the results of vdP and THz-TDS methods, the following equations were used to calculate the relative percent differences on resistance:wing Eq. ) demonstA visual explanation of the correction can be given for sample S3 as an example in Fig. The characteristics of each technique are summarised in Table 41 will become an important part to contribute to the standards development work of the normative bodies, through the initiation of and dissemination of new written standards for the electrical characterisation of graphene based on the Good Practice Guides developed within the project.In conclusion, we introduce a procedure for performing electrical measurements of CVD graphene from macro- to nano-scale using a variety of contact and contactless techniques. The sheet resistance, carrier concentration and mobility were measured using macroscale magneto-transport measurements in the vdP geometry, both in vacuum and ambient conditions to extract the pristine electrical properties of graphene. Upon annealing in vacuum, the carrier concentration has significantly decreased due to desorption of atmospheric molecules (such as water), while the carrier mobility and sheet resistance have significantly increased. The measurements were compared to the sheet resistance maps obtained using microscale THz-TDS mapping, where a weighted average function was applied. For most of the samples, the difference in the sheet resistance as obtained by vdP and THz-TDS was below 5%, demonstrating the good agreement between the different methods. A method for calibrated KPFM measurements of graphene samples is demonstrated, allowing high resolution mapping of the work function on the nanoscale and highlighting the impact of polymer residues on the surface. This work highlights the importance of using a standardized methodology for electrical measurements using a variety of complimentary techniques to obtain a complete understanding of the sample on a variety of lateral scales. The development of these presented metrology and methodologies within the GRACE project3 containing solution. Distilled water was used to clean the film several times after it was transferred onto a quartz substrate. Finally, the PMMA layer was removed by immersion in acetone. In this work we use 4″ CVD graphene sample, which was diced into 10 × 10 mm2 squares. Several diced chips were chosen in random and studied by a number of methods as discussed.The mono-layer graphene growth was carried out in a cold-walled chemical vapour deposition (CVD) reactor (Aixtron BM) using Cu foil as the catalyst. Following the growth, PMMA was spin-coated onto the graphene-covered Cu foil, acting as a sacrificial support layer. The Cu foil was then etched using a FeClSupplementary Information.