Rhynchosciara americana has contributed to studying the biology of invertebrates and the study of the interaction and regulation of genes during biological development. Therefore, this paper aimed to study the R. americana innexin-2 by molecular characterization, analysis of the expression profile and cellular localization. The molecular characterization results confirm that the message is from a gap junction protein and analysis of the expression and cellular localization profile shows that innexin-2 can participate in many physiological processes during the development of R. americana.Gap junctions mediate communication between adjacent cells and are fundamental to the development and homeostasis in multicellular organisms. In invertebrates, gap junctions are formed by transmembrane proteins called innexins. Gap junctions allow the passage of small molecules through an intercellular channel, between a cell and another adjacent cell. The dipteran  The chasame sex . FurtherCommunication between the cytoplasm of one cell with an adjacent cell is essential. Intercellular junctions play a fundamental role in several processes throughout the development of insects, such as during oogenesis, embryogenesis and nervous system development . In animIn vertebrates, the cell junctions known as gap junctions are formed by connexins and pannexins, and the innexins form gap junctions in invertebrates ,11. Studet al. [C. elegans and medicinal leeches [Drosophila melanogaster identified eight genes from the innexins family with several different isoforms [Bombyx mori identified three innexins [Aedes aegypti, six genes encoding innexins were found, and another six genes were found in the stomatogastric ganglion of Cancer borealis [Homarus americanus, 13 gene products from the innexin family were found, six known genes (Homam-Inx1–4 and Homam-Inx6–7) and seven new putative innexin genes (Homam-Inx8–14) [In invertebrates, gap junctions were first identified by Phelan et al. , who sho leeches ,18. In cisoforms ,11,19. Sinnexins ,21. In Aborealis ,23. In HInx8–14) .D. melanogaster, Dm-Inx1 participates in the development of the nervous system [D. melanogaster show that Dm-Inx3 participates in the dorsal closure of the embryo [Anopheles gambiae, disturbances in the expression of Ag-Inx4 result in sterile males, and Ag-Inx7 is essential for the development of the embryonic nervous system [Tribolium castaneum, Tc-Inx7 participates in the blastoderm cellularization process [Innexins have an essential role in the development of insects and other arthropods. In s system . Dm-Inx1s system . Studiese embryo . In Anops system ,29. In T process .Drosophila show that Inx2 acts on calcium transportation between follicular cells (FC) during oogenesis [A. aegypti, Inx2 and Inx7 were the most expressed among innexins, with Inx2 being the most expressed [Aedes albopictus) show the expression of Inx2 in the contact regions of the cells with other adjacent cells and on the free surface of the cells [Scylla paramamosain, Sp-Inx2 has greater expression in cells of the immune system [Periplaneta americana), participating in muscle contractile signalling [Innexin-2 is one of the most studied innexins in insects. Dm-Inx2 participates in epithelial tissue embryonic morphogenesis –34. Studogenesis . Dm-Inx2ogenesis . In the xpressed . Studieshe cells . In Scyle system . Inx2 wagnalling .Rhynchosciara americana (Ra-Inx2) through molecular and morphological techniques. In addition to associate the role of the innexin-2 in the formation of gap junctions during Rhynchosciara americana development evaluating: (i) the nucleotide sequence and the putative protein identifying characteristics of gap junction proteins through bioinformatics analysis, (ii) the mRNA expression profile in germline and somatic lineages and (iii) cellular localization of the protein in R. americana organs and gene localization of the Ra-Inx2 gene in polytene chromosomes.Gap junctions have been observed in several organs and tissues performing the function of cell–cell communication; however, innexin proteins also seem to be important for developing organs and tissues. The main objective of this work was to study the innexin-2 of . 2. 2.1Rhynchosciara americana larvae were collected in the Ubatuba region, state of São Paulo, Brazil, and grown in the laboratory, using the conditions established by Lara et al. [a et al. , with mo. 2.2−1 Proteinase K (Sigma Aldrich). The reaction was incubated for approximately 60 min at 50°C. Then, 1 V phenol : chloroform : isoamyl alcohol (25 : 24 : 1) was added, mixed by inversion and then centrifuged for 5 min at 14 000 rpm. The aqueous phase was transferred to a sterile tube for precipitation and 2.5 V of absolute ethanol and 0.1 V of 3 M sodium acetate pH 5.0 was added. The DNA was resuspended in TE pH 8.0  and treated with RNase, while the RNA was resuspended in H2O RNase Free and treated with DNase  after the nucleic acids were quantified in the NanoDrop ND1000 Spectrophotometer (Thermo Scientific).Nucleic acids were extracted using TMD solution , 10% SDS and 200 mg ml® qPCR Master Mix (Promega), and the reactions were carried out into Corbett Research Rotor Gene 6000 real-time cycler  under the following conditions: 94°C for 5 min, 40 cycles , following melting. The youngest period was used as a calibrator and the results obtained were analysed in the REST 2005 program. Primers used qRaInx2_Left (5′-ACGTGTGCTGCGAAGTTATG-3′) and qRaInx2_Right (5′-AGCGTAAGCAAGCAGAGAGC-3′).The cDNA synthesis was performed using the Improm II Kit - Reverse Transcription System (Promega). The reaction was processed in the Veriti Thermal Cycler (Applied Biosystems). The analysis of the expression profile (quantitative PCR) was done using the GoTaq. 2.3Specific primers for each Ra-Inx2 region are listed in et al. [The protocol established by Siviero et al.  was used. 2.4Analyses of the electropherograms were performed in a Linux operating system using the programs Phred, Phrap, CrossMatch and Consed 17, whose use licenses are free for academic purposes and obtained directly from the authors. The nucleotide sequences were analysed in the BlastX database. The alignments were performed using the ClustalX program (Multiple Sequence Alignment)  and the The ORF-Finder program was used to determine the amino acid sequence. To identify possible transmembrane domains, the TOPCONS program  was used. 2.5R. americana cells, immunofluorescence reactions were performed in the fat body and ovary. After dissecting the larvae, the tissues were transferred to a tube containing 3.7% formaldehyde for 15 min. Then, the tissues were washed twice in PBSA , permeabilized with 1% Triton X-100 for 10 min and washed twice in PBSA. After, the primary antibody  was added and incubated at room temperature for 12 h. After this period, the tissues were washed twice with PBSA for 5 min and then the secondary antibody FITC (Sigma-Aldrich), diluted 1 : 200 in PBSA, was added and incubated for 2 h at room temperature. RNase 10 mg ml−1 was added and incubated for 1 h. The tissues were washed with PBSA and the slides were mounted with propidium iodide and Vecta Shield . The preparations were observed with the laser scanning confocal microscope (LSM510 - Zeiss), and the LSM Image Browser (Zeiss) was used to analyse the images.To identify the location of the Ra-Inx2 protein in . 2.6R. americana fixed in ethanol : acetic acid (3 : 1). Then, the chromosomes were denatured in 0.07 N NaOH for 5 min, then washed three times in 2 X SSC (3.0 M NaCl and 0.3 M sodium citrate pH 7.0), 70% ethanol and absolute ethanol. After drying, the slides received the hybridization mixture containing the probe previously marked with random primer digoxigenin-11-dUTP and denatured by heating. Hybridizations were performed at 58°C with 4 X SSC, for 12 h. After this period, chromosomal preparations were incubated with antibody anti-digoxigenin conjugated to fluorescein, and propidium iodide was used for counterstaining. The preparations were observed using the LSM510 laser scanning confocal microscope  and the LSM Image Browser program was used to analyse the images.The probe used for hybridization was synthesized from the genomic sequence, and primers Inx2_InSitu_Left and Inx2_InSitu_Right were used . The squ. 2.7Tissues were dissected and fixed for 2 h with 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer pH 7.2. The fixed samples were washed in 0.1 M sodium cacodylate buffer pH 7.2 and post-fixed in 1% osmium tetroxide. The tissues were dehydrated in a graded ethanol and propylene oxide series. Resin infiltration was done with a 1 : 1 mixture of propylene oxide and EPON  for 5 h, followed by pure Epon for 5 h. Next, the material was embedded in Epon and polymerized for 48 h. Semi-thin sections were cut using an ultramicrotome and stained with toluidine blue or haematoxylin/eosin. Appropriate regions of the ovaries and fat body were then thin sectioned at 70 and 90 nm and stained with 4% uranyl acetate and a 10% lead citrate solution. The material was analysed with a Jeol 1010 transmission electron microscope at 80 kV.. 3. 3.1a).The genomic sequencing of Ra-Inx2 showed 2874 bp with five exons and four introns. The nucleotides conserved at the ends of the sequence of each intron  were identified; these two pairs of nucleotides being important for the splicing machinery to recognize the sequence to be removed during mRNA processing for mature mRNA formation. The beginning of the sequence is characterized by four exons of approximately 120 to 160 bp and three introns of approximately 60 bp. However, the final part of the sequence is characterized by a long intron of 1606 bp and the last exon of 522 bp. A total ORF of 1077 bp codifying a putative protein of 358 amino acids represents the Ra-Inx2 a.FigureR. americana, such as the fat body , salivary gland (third period), ovary (pupa), testis  and embryo (first and fifth day of embryonic development). All the results obtained from Ra-Inx2 show no alternative splicing in the analysed tissues; all regions of exons are encoded.To check whether alternative splicing occurs during mRNA processing in Ra-Inx2, the region of each exon was amplified from different tissues of b and The putative Ra-Inx2 protein sequence has a conserved domain of 320 nucleotides in the superfamily of innexins. The characteristic regions of gap junction proteins were identified: four transmembrane domains in grey, two red cysteines located in the two extracellular loops and the conserved amino acid residues in green. The amino acid sequence YYQWV, highlighted in pink at the beginning of the second transmembrane domain (TM2), is highly conserved mainly among insects, being considered a signature among innexins , the hydrophobic regions were highlighted in grey corresponding to the four transmembrane domains ; the two extracellular loops (EL1 and EL2) with the two pairs of cysteines highlighted in red, and conserved amino acid residues among the innexins were highlighted in green .ΔG value refers to the distance of each amino acid residue in the transmembrane. The graph highlighted the three intracellular regions in red (inside), corresponding to the amino-terminal region (NT), the intracellular loop (CL) and the CT region. In blue (outside), the two extracellular regions correspond to the two extracellular loops EL1 and EL2, respectively. The four transmembrane domains were also highlighted, with TM1 and TM3 in grey (TM-helix IN → OUT) and TM2 and TM4 in white (TM-helix OUT → IN) (a).The putative protein sequence of Ra-Inx2 was analysed using the Topcons program to confirm the transmembrane structure. The UT → IN) a.Figureb). The prediction of the three-dimensional structure of the Ra-Inx2 protein and alignment with the Dm-Inx2 protein of D. melanogaster was performed using RaptorX program .The probe was synthesized from the genomic sequence, and the images were acquired using a laser scanning confocal microscope. ry gland a,b.Figu. 3.3R. americana was analysed by using qPCR in the following tissues: salivary gland, fat body, ovary, testis and early development embryo. The salivary gland of R. americana is a tissue of large proportions in the larvae [a). In the sixth period, the Ra-Inx2 expression level was increased, being 1.5 times the level of the first period.Gene expression of Ra-Inx2 during the development of e larvae ,55. Physe larvae . The anab).The fat body has a role in the intermediate metabolism in insects, participating in nutrition. In the larval stage, the cells have a flattened shape and are intimately connected to each other. At the end of the larval development, the fat body cells dissociate and reorganize in small clusters during the pupal stage . During figure 7R. americana ovary has characteristics different from the Drosophila in which the oocyte is connected by 15 nurse cells. In R. americana, all ovarian follicles develop synchronously and present only a single giant nurse cell attached to each oocyte. The ovarian follicle develops from the primordial germ cells that differentiate into germ stem cells after some mitotic divisions become germ cells. The last mitotic division of germ cells occurs early in the larval stage, giving rise to two cells with different destinations: the oocyte that enters in meiosis, while the nurse cell undergoes processes of polyploidy and polyteny [c).The polyteny . In the R. americana testis, the developmental process is more dynamic; the stage of growth of the last spermatogonia until the spermatocyte-I is quite long and begins in larvae of approximately 30 days of age. In the pre-meiotic stage, the chromosomes duplicate, and the RNA synthesis is very intense [d).Testis development is different from the ovary in which the oocyte chromosomes are arrested in meiosis and are little active transcriptionally since the transcriptional activity occurs in the nurse and in the FC. In the  intense . Ra-Inx2e).During its development, the embryo is composed of somatic and germinative cell lineages. The expression of Ra-Inx2 occurred at higher levels during the first 4 days of embryonic development. On the second day, the development was more expressive, being 2.1 times greater compared with the first day of development e.. 3.4a).The immunofluorescence reaction with the anti-innexin2 antibody resulted in diffuse labelling on the surface of the ovary of young larvae, suggesting that Ra-Inx2 was in FC. In addition, the positivity (green) was more intense in the fifth period, among the ovarian follicles, similarly to the labelling observed in the third period of the fourth larval stage, localization suggestive of somatic cells. a.Figureb–d.The labelling observed in extra-FC would explain the fact that older pupae present high expression of Ra-Inx2 mRNA, which at first was intriguing because the nurse cell, responsible for RNA synthesis in the oocyte at this age, begins to enter a process of regression and death, and the Ra-Inx2 labelling was detected in FC at this stage. In agreement with these data, intense fluorescence was observed in cells of a tubular structure in the ovary, as illustrated in figure 8b, c illustrates the organization of the R. americana ovary observed in semi-thin sections of transmission electron microscopy preparations. The ovarian follicles are distributed around the duct, and the FC are continuous with the ductal cells. In addition, among the follicles, there are also stromal cells. These cell types are all somatic origin, and data suggest a commitment of these cells to the Ra-Inx2 expression.The organization of the ovarioles around a duct-like structure was evidenced by several techniques, from histological sections to three-dimensional reconstruction of the ovary from images obtained by light-sheet microscopy. Figure 8Rhynchosciara americana ovary preparations did not allow structures resembling gap junctions to be visualized, so the ovaries were analysed for Ra-Inx4 immunofluorescence to compare with Ra-Inx2 result. The immunolocalization data of Ra-Inx4 within the ovariole suggest its association with the germline, different from Ra-Inx2 localization , fat body cells are negative to Ra-Inx2.The immunolocalization of innexin2 in the larval fat body (fifth period of the fourth stage) shows positivity at the cellular boundaries and is more evident at the base of the cells . Interesfigure 10. 3.5R. americana ovary, both from Basile in the 1970s and more recently of the group, had a greater concern with the ovariole and the relation between the nurse cell and oocyte. Revisiting these data and making a broader morphological study now, the existence of other cells in the organs that also express innexins and have gap junctions, is evident. Importantly, mRNA expression profiles relative to whole organs, containing material from different cell types. In the specific case of ovaries and testes, there is a mixture of somatic and germinative cells, reinforcing the importance of new morphological studies.The evidence of the location of gap junctions by immunofluorescence observed under confocal laser scanning microscope pointed to the relevance of visualizing structures corresponding to these types of junctions, under the transmission electron microscope. Previous data focusing on the development of the Cell junctions like gap junctions were observed between somatic cells of the ovaries, mainly between FC, but also between cells at the base of the ovarioles that may correspond to ductal cells. Figures In the fat body of young larvae, the granular endoplasmic reticulum is displaced to the cell boundaries, which makes it challenging to observe junctions . These c. 4R. americana tissues was performed, amplifying by PCR the five exon regions and analysing the data, it was possible to conclude that there is no alternative splicing in the Ra-Inx2 gene. Stebbings et al. [et al. [et al. [Ae. aegypti alternative splicing occurs in Ae-Inx1 and Ae-Inx3, affecting the CT end of these two innexins. Calkins et al. [The Ra-Inx2 genomic DNA sequence analysis resulted in 2874 base pairs, with the presence of four intronic regions corresponding to 1797 base pairs, or 62.5% of the sequence. The search for alternative splicing in different s et al.  show tha [et al.  showed t [et al.  showed ts et al.  also shoThe Ra-Inx2 mRNA encodes a putative protein sequence of 358 amino acids, in agreement with the sequence of innexins characterized in other organisms. The identity of Ra-Inx2 as a gap junction transmembrane protein was confirmed based on the conserved amino acid residues, the two cysteines located in the extracellular loops and the sequences that correspond to the four transmembrane domains. The cysteine residues located in the extracellular loops are essential for anchoring the canal in adjacent cells . In vertet al. [D. melanogaster the EL1 and EL2 loops of Dm-Inx2 have 62 and 63 amino acids, respectively. The results obtained from the analysis of the sequence are in accordance with the results acquired from the TOPCONS program, and in the graph, the value of ΔG represents the distance of each amino acid residue in relation to the membrane [Interestingly, this amino acid is also conserved in the second transmembrane domain of connexins and is associated with the activation of conformational changes in the protein ,66. The et al.  in D. memembrane . Confirmmembrane ,69. The A. gambiae and A. sinensis, proving the values obtained for identity and similarity. Arthropods represent the largest phylum in the animal kingdom, representing approximately 85% of the described animals. The subphylum Crustacea, represented in the tree by Homarus americanus and Cancer borealis, is placed in the same group as insects, confirming the level of conservation among arthropods. Recent studies have confirmed that insects are positioned phylogenetically in the same group as crustaceans [Based on the sequence of amino acids, a phylogenetic tree was constructed comparing the sequence of innexin-2 from other organisms available on GenBank. The tree was built in the MEGA 6 program using the maximum-likelihood method with a bootstrap of 1000 replications, which calculates the percentage of occurrence reliability for a given node in the tree as a percentage. As expected, Ra-Inx2 was grouped with other arthropods, mainly with the dipterans of the Culicidae family, such as staceans ,43,70. Istaceans .R. americana. In parallel during this period, apoptosis occurs in the salivary gland cells occurring during histolysis of this tissue [Spodoptera litura cells and ectopic expression in Spodoptera frugiperda cells promoted cell apoptosis [et al. [R. americana comprised a discrete detection of cell death by TUNEL assay characteristic of the apoptosis process. Active hemichannels may be involved with cell death signalling pathways or inhibiting cell survival pathways [2+ increasing the mitochondrial outer membrane permeabilization (MOMP). MOMP is a factor that induces apoptosis, causing the release of cytochrome C and activation of the caspase pathway [R. americana, possibly participating in the remodelling of the fat body and the histolysis of the salivary gland.Gene expression indicated that a higher expression of Ra-Inx2 occurs in the salivary gland in the sixth period of the development of s tissue . Howeverpoptosis . Connexipoptosis . In the  [et al.  the levepathways . The act pathway . The Ra-et al. [D. melanogaster ovary of Dm-Inx1, Dm-Inx2, Dm-Inx3, Dm-Inx4 and Dm-Inx7. Other studies show that Dm-Inx2 was co-localized with Dm-Inx3 in somatic cells and co-localized with Dm-Inx4 in a germ cell line [Drosophila ovarian follicles resulted in follicles without ovaries, small ovaries and few follicles [D. melanogaster ovaries demonstrate that Inx2 and Inx3 act on border cells regulating microtubules, with Inx4 having a similar role in the oocyte. The RNAi knockdown of these innexins destabilizes the microtubules causing morphological changes in the border cells and oocyte [R. americana, Ra-Inx2 showed an expression pattern found in ovaries, and this pattern was also identified by Hong et al. [B. mori. The expression of Bm-Inx2, Bm-Inx3 and Bm-Inx4 in the testis was very similar to the expression found in the ovary during the larval, pre-pupal and pupal phase, with Bm-Inx4 being the most expressed innexin [Ae. aegypti, the expression of Ae-Inx2 was considered strong in the testis [R. americana. In the embryo, the expression of Ra-Inx2 remained constant throughout the 1–4 days of development; this expression profile was also observed by Hong et al. [B. mori, and the expression of Bm- Inx2 and Bm-Inx3 maintained the same expression profile throughout embryonic development, different from Bm-Inx4 that was expressed only on the first day of development [D. melanogaster, the innexins Dm-Inx1, Dm-Inx2 and Dm-Inx3 participate in the dorsal development of the embryo [et al. [Ra-Inx2 expression increased throughout the fourth stage of development in the ovary, which may be associated with the fact that the ovarian follicle increases considerably in size from the third period. During this period, the nurse cell goes through endo-replicative cycles, occurring in the polyploidy process . Howeveret al.  showed tell line . Dm-Inx2ollicles . Recent d oocyte . In the g et al.  in B. mo innexin ,21. In Ae testis . In the g et al.  in B. moelopment ,21. In De embryo . Holcrof [et al.  demonstr [et al. ,34.in situ hybridization, a somatic chromosome, differently from Drosophila. Dm-Inx2 gene is located on X chromosome of D. melanogaster in 6E4 region, which includes a cluster of innexins genes, such as Dm-Inx1 and Dm-Inx7 [D. melanogaster ovary, the location of Dm-Inx2 was also preferentially located in somatic cells. According to Bohrmann & Zimmermann [et al. [The Ra-Inx2 gene is in region 17 of chromosome A by  Dm-Inx7 . To showmmermann , the Dm- [et al.  show thaTake all together, gap junction channels are fundamental for the physiology of multicellular organisms. In particular, innexin-2 has become one of the most studied innexins among different organisms due to its characteristic of acting in different tissues of somatic and germinative lineages.


Multicomponent exercise that includes both resistance and aerobic training is recommended to prevent loss of bone mineral density (BMD) in postmenopausal women. However, optimal training frequency has not been determined. Sixty-three non-osteoporotic sedentary women ages 60y and older were randomized to one of three exercise groups for sixteen weeks: 1) one resistance and one aerobic session per week, 2) two resistance and two aerobic sessions per week, or 3) three resistance and three aerobic sessions per week. Resistance exercise included supervised sessions on weight machines, and aerobic exercise was treadmill walking. BMD of the hip and lumbar spine (L1-L4) was assessed by dual energy X-ray absorptiometry , and z scores were calculated from a reference population adjusted for age and sex. Among the total cohort with BMD measurements at week 16 , z scores improved for the trochanter, Ward’s triangle, total hip, L1 and L4. Within-group comparisons showed improvement at the trochanter, total hip, and L1 for group 2 only, while only group 1 demonstrated an increase at L4 . However, no time-by-group interactions were observed. Sixteen weeks of combined resistance and aerobic training is effective for improving BMD of older adult women. Results suggest training frequency of two sessions per week may be optimal. Postmenopausal women should be encouraged to do aerobic exercise such as walking plus resistance training at least once weekly to prevent osteoporosis.


Culture-independent studies have revealed that chronic lung infections in persons with cystic fibrosis (pwCF) are rarely limited to one microbial species. Interactions among bacterial members of these polymicrobial communities in the airways of pwCF have been reported to modulate clinically relevant phenotypes. in silico approaches can help build clinically relevant in vitro models of polymicrobial communities that can in turn be used to experimentally test and validate computationally generated hypotheses. Furthermore, we posit that combining computational and experimental approaches will enhance our understanding of mechanisms that drive microbial community function and identify new therapeutics to target polymicrobial infections.Culture-independent studies have revealed that chronic lung infections in persons with cystic fibrosis (pwCF) are rarely limited to one microbial species. Interactions among bacterial members of these polymicrobial communities in the airways of pwCF have been reported to modulate clinically relevant phenotypes. Furthermore, it is clear that a single polymicrobial community in the context of CF airway infections cannot explain the diversity of clinical outcomes. While large 16S rRNA gene-based studies have allowed us to gain insight into the microbial composition and predicted functional capacities of communities found in the CF lung, here we argue that  CFTR, leading to defective chloride secretion and reduced airway surface liquid hydration .Cystic fibrosis (CF) lung disease results from a cascade of events that begin with mutations in a single gene coding for an ion channel, ydration . Loss ofydration . As descPseudomonas aeruginosa, Staphylococcus aureus, Streptococcus spp., and anaerobic species, are capable of engaging in multiple interactions impacting clinically relevant features such as virulence, persistent colonization of the airways, and antimicrobial recalcitrance  negatively affect respiratory function of pwCF when they cocolonize the airways compared to when they are present alone  that would explain the consequences of polymicrobial interactions in the context of the CF airway, as well as the impact of community interactions on antibiotic responsiveness and disease outcomes? Having such model communities could provide tremendous benefits in terms of (i) focusing efforts of the research community on understanding mechanisms of microbial interactions, (ii) exploring how such interactions drive persistence, antibiotic recalcitrance, and virulence, and (iii) serving as a platform to discover novel antimicrobials that are effective against such complex communities. However, one of the major hurdles in defining such a community is the existing interpatient heterogeneity in microbial community composition. For example, in one of the earliest culture-independent CF studies examining microbial communities in the lungs of pwCF, Rogers and colleagues observed that the 16S rRNA gene-based detection of key CF pathogens, such as o person . Subsequo person –25. Simi person –. They redividual . Furtherdividual , the impdividual , 27, 28.dividual , 30. ThuHow then, can one identify and select a model community to study in the context of polymicrobial infections existing in the CF airway? With the multiple lines of evidence pointing at the heterogeneous microbial communities existing between pwCF, we would argue that this is the wrong question to ask. Instead, one should ask: are there a limited number of communities that would encompass most of the microbial diversity encountered in the CF airway? In an attempt to address this open question, we present two different analyses below.First, previous work from our team addressed this question using 16S rRNA gene data from a small number of pwCF. We analyzed sputum samples from 35 pwCF, either undergoing a clinical flaring of disease  that required hospitalization  could impact a polymicrobial biofilm community composed of P. aeruginosa, S. aureus, Streptococcus anginosus, Achromobacter xylosoxidans, R. mucilaginosa, and Gemella haemolysans compared to their monospecies counterparts. Interestingly, the authors observed that the antibiotic susceptibility of bacterial species grown as a community was similar to when they were grown as pure culture biofilms. This finding was surprising given previous studies showing the complex response of mixed communities to antibiotic treatment  collected from six pwCF has recently been published , which often require hospitalization and intravenous antibiotic treatment. Thus, it might be possible to note changes in metabolotype across the disease cycle, from exacerbation to treatment to clinical recovery, versus the baseline state of those airway communities. These temporal changes in metabolotype may call for  distinct therapeutic strategies for effective treatment of the airway infections. Indeed, a recent report examining the metabolomic profile of a large number of longitudinal sputum samples (ublished . In thisLast, an important caveat to most of the microbiome studies published so far is the strong focus on bacteria; most reports have overlooked the presence of fungi and viruses and their impact on CF airway disease , 43, 44.


Thus the interaction between the nanosheets and the layers of nanosheets is weak and the friction coefficient is low (0.06–0.07). While the friction coefficient increases to 0.14–0.15 in a nitrogen atmosphere due to the interaction of defects generated in the sliding process, the nitrogen molecules with lone pair electrons can only make the nanosheets passive to a certain degree, thus the ordered slip structure is destroyed and friction is higher. This work reveals the influence of environmental molecules on the macroscale tribological performances of graphene and its effect on the microstructure at the sliding interface, which could shed light on the lubricating performance of graphene in environmental atmospheres and help us to understand the tribological behaviors of graphite at the macroscale.This study investigated the friction behavior of graphene in air and nitrogen atmosphere environments. The microstructural evolution caused by the variation of atmosphere environments and its effect on the friction coefficient of the graphene is explored. It is demonstrated that graphene can exhibit excellent lubricating properties both in air and nitrogen atmosphere environments. In air, a highly ordered layer-by-layer slip structure can be formed at the sliding interface. Oxygen and H Reducing friction and wear losses has great value for the moving components of sliding conditions . As the Microscale tribological studies of lubricating materials, especially the studies of structure could guide understanding of the origin of the tribological properties and macroscale lubricating mechanism . As the Based on this, the macroscale tribological properties of graphene in air and nitrogen atmospheres were explored, and the response behaviors to the surrounding atmospheres were studied. Then the corresponding microstructure of the sliding interface and the morphologies of the frictional pair were characterized by the transmission electron microscope (TEM) and Raman spectra as well as optical image to investigate the influence of atmosphere environments on the microstructure and the effect on the macroscale frictional behavior of graphene. The research of the structural evolution induced by the surrounding atmospheres and their influence on the friction coefficient during the atmosphere change helps reveal the macroscale tribological performance of graphene and thus furthers understanding of the lubricating mechanism of graphite at the macroscale, which could be significant for providing guidance for graphene (graphite) to be used as a solid lubricant at the macroscale.Graphene powder was purchased from Nanjing Jicang Nano Technology Company Limited , and the graphene nanosheets are prepared by mechanical exfoliation. Analytical grade volatile alcohol  was commercially obtained. The purchased graphene powder and volatile alcohol without any chemical treatment were chosen to explore the inherent properties of pure graphene. The original structure of graphene is shown in A pin-on-disk CSM tribometer  was used to conduct the frictional tests of the graphene coating. The counterpart ball commercially purchased AISI52100 steel ball  was chosen. The friction experiments were performed at room temperature (23 ± 2°C), and the atmosphere is controlled by the chamber that the tribometer carried itself , and the operating voltage was 40 KV and the operating current was 40 mA, and copper target (λ ≈ 0.154 nm) was used. The Raman spectra of graphene surface, wear tracks, and wear scars of the counterpart ball were analyzed by Raman spectroscopy  using a wavelength of 532 nm (2.3 eV) lase through the 50X objective lens. The optical images of the original surface, wear scars, and wear tracks of graphene coating were investigated by optical microscope . The graphene powder was dispersed in ethanol and ultrasonic for approximately 30 min, and then the dispersions were transferred to the Cu grid to observe the morphologies and structures. The structural evolution of the sliding interfaces after 30 min rubbing was transferred to the Cu grid to observe their structures and morphologies. The morphology of graphene powder and sliding interface was characterized by Transmission Electron Microscopy  and high resolution transmission electron microscopy (HRTEM). The lateral size of the graphene nanosheets was tested by the Zetasizer Nano ZS laser dynamic scattering instrument .D/IG is about 0.06). The graphene nanosheet is about 4–5 layers, and d-spacing is about 0.34 nm . The uniform transferred films  can passivate the edge dangling bonds of graphene and the defects generated during the friction process. Thus, the interaction between the interlayer and the lamellae is weak and the friction coefficient is low. Inert gas molecules (such as nitrogen) possess the lone pair electrons that could interact with the π orbital of graphene and play a role in passivation to a certain degree, rendering the graphene to exhibit excellent macroscale lubricating properties.The work above shows that gas molecules in different atmospheres have different effects on the microstructure of the sliding contact interface of the graphene during the friction process so that graphene exhibits different friction behaviors. The friction coefficient of graphene depends on the interaction between its layers and the nanosheets. Active gas molecules . In particular, the active gas molecules in the air can passivate the defects and edge dangling bonds, and H


Involving the end-users of scientific research  in setting research priorities is important to formulate research questions that truly make a difference and are in tune with the needs of patients. We therefore aimed to generate a national research agenda for Juvenile Idiopathic Arthritis (JIA) together with patients, their caregivers and healthcare professionals through conducting a nationwide survey among these stakeholders.The James Lind Alliance method was used, tailored with additional focus groups held to involve younger patients. First, research questions were gathered through an online and hardcopy survey. The received questions that were in scope were summarised and a literature search was performed to verify that questions were unanswered. Questions were ranked in the interim survey, and the final top 10 was chosen during a prioritisation workshop.Two hundred and seventy-eight respondents submitted 604 questions, of which 519 were in scope. Of these 604 questions, 81 were generated in the focus groups with younger children. The questions were summarised into 53 summary questions. An evidence checking process verified that all questions were unanswered. A total of 303 respondents prioritised the questions in the interim survey. Focus groups with children generated a top 5 of their most important questions. Combining this top 5 with the top 10s of patients, carers, and clinicians led to a top 21. Out of these, the top 10 research priorities were chosen during a final workshop. Research into pain and fatigue, personalised treatment strategies and aetiology were ranked high in the Top 10.Through this study, the top 10 research priorities for JIA of patients, their caregivers and clinicians were identified to inform researchers and research funders of the research topics that matter most to them. The top priority involves the treatment and mechanisms behind persisting pain and fatigue when the disease is in remission. Juvenile Idiopathic Arthritis (JIA) is a heterogeneous disease that is characterised by the occurrence of arthritis of unknown origin lasting for more than 6 weeks, with an onset before the age of 16 years. It is the most common chronic rheumatic disease in children . NeverthWhile the importance is widely acknowledged , still lThe JLA is a non-profit initiative, founded in 2004 in the United Kingdom to bring patients, carers , and clinicians together in Priority Setting Partnerships (PSPs) to set research priorities , 13. TheThe Medical Ethical Committee of University Medical Centre Utrecht confirmed that this study was exempted from the Medical Research Involving Humans Act (WMO) (METC protocol number 18–721/C).This PSP was conducted in the Netherlands. The full set-up is demonstrated in Fig. n = 4), parents of JIA patients (n = 3), paediatric rheumatologists (n = 3), an ophthalmologist (n = 1), physical therapists (n = 2) and a nurse practitioner (n = 1). Clinicians were recruited from 7 academic centres in the Netherlands. They were selected to represent all different professions and all geographic regions of the Netherlands. Most of them were members of the abovementioned organisations. Our JLA advisor (KC) had an advisory role throughout the whole process, while the steering group had the authority to make all important decisions within the framework and guiding principles of the JLA.The PSP was initiated by two Dutch patient associations for JIA (the Dutch JIA patient and parent organisation (JVN) and Youth-R-Well.com (YRW)), and two Dutch professional organisations (the Dutch Society for Paediatric Rheumatology (NVKR), and the Dutch Health Professionals in Paediatric Rheumatology (NHPKR)). An expert steering group was assembled to oversee all phases of the project. This group consisted of young adult JIA patients . Patients and parents were recruited to participate via patient organisations , and via flyers distributed at all outpatient clinics for paediatric rheumatology in the Netherlands. Clinicians were recruited via professional organisations’ newsletters and flyers were sent to them. Demographic details were analysed periodically to strive for equal representation of age, education level and region. Recent JIA guidelines were also searched for research recommendations. Extracted recommendations were added to the question database. Focus groups and small (group-) interviews were held with children (aimed 10–13 years) in an exploratory, conversation-like manner, guided by an expert in child participation . In-depth results of the focus groups will be published separately .All questions were reviewed and assessed whether they were in scope. Questions that were identified out of scope were put on a separate list which was agreed upon in a steering group meeting. Next, the included questions were grouped into categories and keywords were allocated to them, to ease the process of summarising. In a face-to-face meeting, the steering group summarised the questions into summary questions. A search in PubMed and PsycINFO (restricted to the last 10 years) was conducted to ensure that the question was unanswered. Publications were found to be relevant if they included systematic reviews, meta-analyses, randomised control trials (RCTs) or large representative cohort studies following the JLA guidelines. Next, the steering group provided expert opinion on the articles that were retrieved from this search and made the final decision about whether or not the question could be identified as already answered.Here, the goal was to make a shortlist of 20–25 questions for the final workshop. Again, a survey was designed in which participants could choose their Top 10 (October–December 2019). Participants were recruited in the same way as during the first survey. Furthermore, participants of the first survey who left their contact details were e-mailed directly. Younger children were asked to rank their top 5 during focus groups that were set up as small priority setting workshops. Three separate top 10s, for patients, carers and clinicians, were determined. Together with the top 5 of the younger children, a final shortlist was set – they all had an equal weight. This shortlist was taken to the final workshop.Participants of the workshop were invited from the steering group, through professional connections and through the interim survey (people who left their contact details were approached) to be inclusive of the whole JIA community. Prior to the workshop, participants were asked to individually prioritise the shortlist of questions. Through three rounds of prioritisation in both smaller and larger groups, chaired by trained facilitators using an adapted nominal group technique, the final top 10 was agreed (February 2020). Several observers were invited from a large patient advocacy organisation, an insurance company, the Dutch Society for Paediatrics (NVK) and the Dutch Arthritis Foundation. They did not take part in the discussions, but were present to observe the process, become inspired and learn from the discussions.An independent ethical process evaluation was conducted by a bio-ethicist (KJ) in order to evaluate (the inclusiveness of) the priority setting process. The bio-ethicist was present during all steering group meetings, the final workshop, and was included in all correspondence regarding the research agenda. In-depth results of this evaluation will be published separately .During the first phase of the process, a total of 604 questions were gathered. Of these 604, 506 were submitted by 278 respondents through the survey: 141 patients (50.7%), 88 carers (31.6%) and 49 health care professionals (17.6%). Demographic data are summarised in Table Of the 604 submitted questions, 519 were determined to be in scope. Out of scope questions mostly related to logistics of care  or personal situations (e.g. “Which type of JIA do I have?”) and could not be answered by research. During a face-to-face meeting, the steering group summarized the remaining questions into 53 summary questions.An elaborate search in PubMed and PsycInfo showed that none of the 53 questions was completely answered. A full list of all the submitted questions, the 53 summary questions, our search strategy for evidence checking and the critical appraisal of the studies has been published online .Three hundred and three people chose their top 10 from the 53 questions: 125 patients (41.3%), 136 carers (44.9%) and 42 clinicians (13.9%). Two focus groups with seven and nine children respectively , and a subsequent group priority setting discussion attended by six girls and five boys from the focus groups, resulted in a top 5 of their most important questions .The 35 questions in the respective top 10s of patients, carers, clinicians, and the top 5 of younger children showed considerable overlap. One question – on fatigue – was prioritised by all 4 groups. Four questions were ranked in three top 10s. This resulted in a combined top 21. Table n = 3), an ophthalmologist (n = 1), physical therapists (n = 2), nurses (n = 3), and a psychologist (n = 1)). Clinicians were recruited from all academic centres in the Netherlands. The results of two rounds of discussions and ranking in three mixed groups were combined, and discussed further in a final session. The participants chose to merge the two summary questions on personalised medicine and strategies to taper off medication  were selected. For clinicians it was 80%. For the focus groups 2 out of 5 questions (40%) were part of the final top 10. Importantly, question 9 of the final top 10 made it into the list because a young adult patient advocated for it during the final discussion, using the argument that it was the children’s top priority. Evaluations from the participants of the final workshop were very positive: patients, carers and clinicians felt like their voice was heard, that all voices mattered equally, and that the atmosphere was very positive.This project has brought patients, caregivers and clinicians together in creating a research agenda for JIA using the JLA method. This is one of the first times a PSP has integrated the JLA approach with additional focus groups with children to ensure involvement of paediatric patients of all age categories. We found that the number one research priority involved the cause and care of pain and fatigue when the disease was in remission. This is in line with a qualitative study by Bromberg et al. that finds self-reported pain and fatigue are highly common in children with JIA despite advances in treatment strategies . It is aMoreover, our study exposed other important research areas such as personalised treatment strategies, JIA-associated uveitis, nutrition, long-term effects of drugs taken for JIA and sports/exercise that were not directly featured in other studies. This may be due to the wide scope we defined for this project, as well as the easily accessible nature of the JLA method that may have led to the collection of more widespread themes. The fact that the questions do not only cover the healthcare setting but also comprise more (psycho) social and educational issues suggests funding for these topics can also be sought from psychological, social and educational funding bodies.One major strength of our study is the use of the systematic and transparent JLA method, which enhances the validity of our results. Furthermore, our efforts to include younger children is a strength. We conducted focus groups during the two phases of the process where information was gathered, such that they truly had their say in determining the most important topics. Two questions of the children’s Top 5 made it to the final Top 10 , that were not prioritized in the online parent group. This may have been related to their own educational background.In this light, it is important to realise that participants of the final workshop consisted of a group of (predominantly) white, articulate, higher educated people, despite explicit efforts to include people with lower educational backgrounds and other ethnicities. This may have influenced which questions made it to the Top 10. This is a typical tendency in survey research and was also observed in other PSPs . Future Formally, this Top 10 was generated in the Netherlands, but we expect it also holds for other western countries. Now that the research agenda has been set, it is important that it is now indeed implemented with research funders and researchers. To achieve this, we attempted to inform the whole JIA research community in the Netherlands of this prioritisation exercise from the start, and some of the research groups made the commitment to truly incorporate the Top 10 in the long-term vision of their research lines. In addition, we have organised workshops with researchers, patients, parents and clinicians to turn the relatively broad questions into research proposals that can be presented to funding agencies. These workshops took place in the fall of 2020. With these workshops, we hope to gather a group of people that is truly committed to finding answers to the research questions of the Top 10.In short, we systematically and transparently generated a research agenda for JIA, confirmed as evidence uncertainties and regarded important by children and young people with JIA, their parents/caregivers and the clinicians caring for them. This is a vital resource that conveys a clear message to both government funders and charitable agencies, about which research topics should have priority. The next step is to turn the questions into research proposals. Workshops are currently being organised to this end.


The analysis of the race model showed that AVI was decreased and delayed with the addition of auditory sustained attention (no_load > load_1 > load_2 > load_3 > load_4) for both older and younger adults. In addition, AVI was lower and more delayed in older adults than in younger adults in all attentional load conditions. These results suggested that auditory sustained attentional load decreased AVI and that AVI was reduced in older adults.The modulation of attentional load on the perception of auditory and visual information has been widely reported; however, whether attentional load alters audiovisual integration (AVI) has seldom been investigated. Here, to explore the effect of sustained auditory attentional load on AVI and the effects of aging, nineteen older and 20 younger adults performed an AV discrimination task with a rapid serial auditory presentation task competing for attentional resources. The results showed that responses to audiovisual stimuli were significantly faster than those to auditory and visual stimuli (AV > V ≥ A, all  Individuals are constantly exposed to information from different sensory sources; however, they can select to attend to some of the available information and suppress useless information to identify outside events. The procedure that integrates information from different sensory sources is called multisensory integration , 2. StudTo study the effect of visual attentional load on AVI, Alsius et al. instructed participants to conduct the classic McGurk test under low (single task) and high  attentional load conditions, and both behavioral and EEG results showed decreased AVI under high attentional load conditions than under low attentional load conditions , 11. SimWahn and König reported that distinctions between or sharing of auditory attention and visual attention are task dependent ; in partIn addition to attentional load, aging is also an important factor that alters an individual's perception of auditory information and visual information and includes decreased visual sensitivity  and incrTwenty healthy older adults and 20 young adults were recruited to participate in the current study. All of the older adults were recruited from Huaxi University Town of Guiyang City, and all of the younger adults were college students at Guizhou University of Traditional Chinese Medicine. All participants provided written informed consent for the procedure, which had been previously approved by the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. All participants were free of neurological diseases, had normal hearing, had normal or corrected-to-normal vision, were right-handed, and were naive to the purpose of the experiment. Participants were excluded if their Mini-Mental State Examination (MMSE) scores were greater than 2.5 SDs from the mean for their age and education level . AdditioSimilar to our previous study , two tasFor the AV discrimination task, the auditory nontarget was a 1000 Hz sinusoidal tone, and the auditory target was white noise. The visual nontarget was a black and white checkerboard image , and the visual target was a B/W checkerboard image with two black dots contained within each white checkerboard . The audFor the RSAP task , the audThe stimulus presentations and data collection were controlled using MATLAB R2013b . Subjects were performed the experiment in a dimly lit and sound-attenuated room . To fully understand alterations in AVI with the addition of attentional load, AVI was assessed in five different attentional load conditions. In all attentional load conditions, the AV discrimination task was identical , but theFor the AV discrimination task, a fixation cross was presented for 3000 ms, and then, the A, V, and AV stimuli were randomly presented with a random interstimulus interval (ISI) of 2000-2500 ms . The parFor RSAP task, 90 characters with 10 times for each character were randomly presented with a 2000-2500 ms interstimulus interval. In the no-attentional-load condition, the RSAP task was not presented, but it was presented simultaneously with the AV discrimination task in all other attentional load conditions. In the attentional load_1 condition, the participants were instructed to respond only to the target in the AV discrimination task but to withhold responses associated with the RSAP task. In the attentional load_2 condition, the participants were instructed to respond to the target in the AV discrimination task by pressing the left button of the mouse and to the target  in the RSAP task by pressing the right button of the mouse. In the attentional load_3 condition, the participants were instructed to respond to the target in the AV discrimination task by pressing the left button of the mouse and to the target  in the RSAP task by pressing the right button of the mouse. In the attentional load_4 condition, the participants were instructed to respond to the target in the AV discrimination task by pressing the left button of the mouse and to the target  in the RSAP task by pressing the right button of the mouse. During the experiment, the participants were instructed to treat the AV discrimination task and the RSAP task equally, and the five attentional load sessions were conducted in a random order for each participant. In the attentional load_2, load_3, and load_4 conditions, when the target of AV discrimination task and the target of RSAP task were presented simultaneously, the participants were instructed to press the left and right button of the mouse simultaneously.group  × 5attentional load  × 3stimulus  ANOVA (Greenhouse-Geisser corrections with corrected degrees of freedom). The statistical significance level was set at p ≤ 0.05, and the effect size estimates, ηp2, are also reported.The accuracy and response times  were computed separately for each subject under each condition, and then, the data were submitted to a 2P(AV)] will never exceed the probability of the race model [P(A) + P(V)–P(A) × P(V)]. The coactivation model hypothesizes that the A stimulus and the V stimulus integrate together to achieve the criterion and trigger the response. Therefore, if the probability of the response to AV was significantly faster than that in the race model, it was assumed that the race model was violated and the coactivation model was supported, that is, AVI occurred. P(A), P(V), and P(AV) denote the probability of responding within a given time during auditory trials, visual trials, and audiovisual trials, respectively. To compare the difference in the amount of AVI in various attentional load conditions, a difference probability curve was generated by subtracting a subject's race model CDF from his/her AV CDF in each 10 ms bin , revealing higher accuracy by the younger adults than by the older adults, and a significant main effect of attentional load , revealing the highest accuracy in the no-attentional-load condition (no > load_1 = load_2 > load_3 > load_4), which indicated that the establishment of attentional load was reasonable. In addition, a significant main effect of stimulus  revealed higher accuracy when responding to AV stimuli than when responding to A or V stimuli , which indicated response facilitation to AV stimuli.The accuracy of one older participant was 54% and was not included in further analysis. For other 19 older and 20 younger adults, the excluded trials were less than 5% in all attentional load conditions, and their accuracy and RTs were calculated separately  and thengroup  × 5attentional load  × 3stimulus  ANOVA on RTs showed a significant main effect of group , attentional load , and stimulus . The analysis revealed faster responses by younger adults than by older adults, faster responses in the no-attentional-load condition than in other attentional load conditions (no ≥ load_1 > load_2 ≥ load_3 > load_4), and faster responses to the AV stimuli than the A and V stimuli . There was a significant interaction between attentional load and group . The post hoc analysis of pairwise comparison showed that responses by the younger adults were faster than those by the older adults in all attentional load conditions . Responses were faster in the attentional load_3 condition than in the attentional load_4 condition for older adults (no = load_1 > load_2 = load_3 ≥ load_4) but not for younger adults (no = load_1 > load_2 = load_3 = load_4). Additionally, the interaction between attentional load and stimulus was also significant . The post hoc analysis of pairwise comparison showed faster responses to the V stimuli than to the A stimuli in the load_2, load_3, and load_4 conditions  but not in the no-attentional-load condition and attentional load_1 condition (AV > V = A). The pairwise comparison analysis across stimuli revealed faster responses to the V stimuli in the attentional load_3 condition than in the attentional load_4 condition (no = load_1 > load_2 = load_3 > load_4) but not when responding to the A and AV stimuli (no = load_1 > load_2 = load_3 = load_4).The 2As shown in Significant AVI was found in all attentional load conditions for both older and younger adults, except in the attentional load_4 condition for older adults , Table 2The peak latency was delayed in the older adults compared with the younger adults in all attentional load conditions, with latencies of 460 ms vs. 370 ms, 470 ms vs. 440 ms, 480 ms vs. 390 ms, and 510 ms vs. 450 ms for the no-attentional-load, attentional load_1, attentional load_2, and attentional load_3 conditions, respectively. The peak latency was 450 ms for the younger adults in the attentional load_4 condition. The time window was also delayed in the older adults compared with the younger adults in all attentional load conditions, with time windows of 350-500 ms vs. 250-450 ms, 400-500 ms vs. 300-510 ms, 450-490 ms vs. 320-450 ms, and 500-550 ms vs. 350-500 ms for the no-attentional-load, attentional load_1, attentional load_2, and attentional load_3 conditions, respectively. The time window was 380-500 ms for the younger adults in the attentional load_4 condition. These results suggested that AVI was delayed with the addition of attentional load and was more delayed for the older adults compared with the younger adults in all attentional load conditions.The aim of the present study was to investigate how sustained auditory attention affects AVI and the effect of aging. The results showed that AVI was decreased and delayed with the addition of sustained auditory attentional load. In addition, AVI was lower and more delayed in the older adults than in the younger adults under all sustained auditory attentional loads.Consistent with some previous studies –12, AVI time-window-of-integration model,” which presumed that the integration of auditory information and visual information included two stages: early afferent processing (first stage) and converging subprocesses (second stage) [With the increase in attentional load, AVI was delayed. Responses were slower in conditions with distractors than in conditions without distractors , 50, so d stage) , 54. TheAVI was lower in the older adults than in the younger adults in attentional load conditions. An enhanced AVI was found in studies by Laurienti et al. , 40 and


Insoluble inclusions of phosphorylated TDP-43 occur in disease-affected neurons of most patients with amyotrophic lateral sclerosis (ALS) and about half of patients with frontotemporal lobar degeneration (FTLD-TDP). Phosphorylated TDP-43 potentiates a number of neurotoxic effects including reduced liquid–liquid phase separation dynamicity, changes in splicing, cytoplasmic mislocalization, and aggregation. Accumulating evidence suggests a balance of kinase and phosphatase activities control TDP-43 phosphorylation. Dysregulation of these processes may lead to an increase in phosphorylated TDP-43, ultimately contributing to neurotoxicity and neurodegeneration in disease. Here we summarize the evolving understanding of major regulators of TDP-43 phosphorylation as well as downstream consequences of their activities. Interventions restoring kinase and phosphatase balance may be a generalizable therapeutic strategy for all TDP-43 proteinopathies including ALS and FTLD-TDP. TARDBP gene, is a nucleic acid binding protein originally identified as a transcriptional repressor of the human immunodeficiency virus type 1 (HIV-1) trans-activator response (TAR) long terminal repeats [TARDBP gene cause some familial cases of ALS, indicating pathological TDP-43 is not only a hallmark of disease, but also a driver of disease [The TAR DNA-binding protein TDP-43), encoded by the highly conserved  repeats ). The di, encoded repeats . Mutatio disease .Pathological TDP-43 accumulates in the disease-affected neurons of ~ 97% of ALS patients and ~ 50% of FLTD patients , phosphoTDP-43 pathology can accumulate in non-demented aged individuals , althougTDP-43 contains protein domains consistent with its known functions, including an N-terminus with a nuclear localization signal (NLS), two RNA recognition motifs RRM1 and RRM2), and a C-terminus glycine-rich low-complexity domain  mRNA levels correlate with phosphorylated TDP-43 in ALS patient tissue and overexpression of CSNK1E in iPSC-derived motor neuron lines promotes TDP-43 cytoplasmic accumulation [While phosphorylation of TDP-43 could represent an aberrant cellular process during disease, several lines of evidence suggest this is a regulated event. To date, five kinases have been shown to directly phosphorylate TDP-43: casein kinases 1 and 2 (CK1 and CK2), cell division cycle 7 (CDC7), and tau tubulin kinases 1 and 2 (TTBK1 and TTBK2)  dramatically reduces the ability of TDP-43 to polymerize. Furthermore, S48E slows TDP-43 phase separation and results in more soluble phase separated TDP-43 nuclear assembles . A recenDrosophila does not alter TDP-43 aggregation. Various combinations of phosphomimetic mutations S379E, S403E, S404E, S409E, and S410E, or expression of the kinase CK2α, are able to reduce the number of aggregates in cells. In these studies, S409/410 phosphorylation occurs after aggregation suggesting TDP-43 phosphorylation may be compensatory, delaying the formation of larger aggregates by electrostatic repulsion at the expense of decreasing TDP-43 turnover [However, not all studies suggest phosphorylation promotes aggregation. In HEK-293 cells, TDP-43 15 kDa and 25 kDa fragments with phosphomimetic mutations S409D/S410D show 60% less aggregation compared to wild type fragments suggesting a possible compensatory role for TDP-43 phosphorylation. Non-phosphorylatable mutations S409A/S410A also reduce aggregation, but only by 15% . Likewisturnover .While the timing and subcellular localization of TDP-43 phosphorylation in disease remains largely unexplored, two recent studies support S409/410 phosphorylation occurring after aggregate formation. To drive aggregation, an optogenetic TDP-43 construct that undergoes oligomerization after blue light exposure (optoTDP43) was expressed in HEK-293 cells or ReNcell cortical neuron cultures. After induction, optoTDP43 forms stable neurotoxic inclusions, a subset of which are phosphorylated . AnotherC. elegans models, neuronal expression of human TDP-43 with fALS mutations G290A or M337V kills motor neurons. Prevention of S409/410 phosphorylation with non-phosphorylatable mutations S409A/S410A improves locomotor dysfunction [doubletime, the fly homolog of CSNK1E, in the Drosophila eye increases S409/410 phosphorylation and the formation of toxic oligomeric species. Cytotoxicity of TDP-43 oligomers is also enhanced in SY5Y cells when treated with a recombinant rat CK1 [In function . Similar rat CK1 .Taken together, the referenced studies demonstrate that TDP-43 phosphorylation influences normal TDP-43 functions including RNA binding, alternative splicing, localization, phase separation, aggregation, and clearance. Yet, many questions remain with regard to the sub-cellular localization, timing in disease progression, potential for additional phosphorylation priming, and full functional impact of site-specific phosphorylation. For example, phospho-specific antibodies raised against TDP-43 phosphorylated at S369 and S379 immunostain insoluble cytoplasmic inclusions in ALS/FTLD-TDP patient brain tissue, but the functional impact of phosphorylation at these sites on TDP-43 is unknown. Disease-causing missense mutations introducing or deleting TDP-43 phosphorylation sites also point to important roles for phosphorylation in health and disease Fig. . It remaThe kinases and phosphatases that regulate TDP-43 phosphorylation are possible therapeutic targets in TDP-43 proteinopathies. However, a single primary TDP-43 kinase has not been determined in neurons; given possible overlap or redundancy in TDP-43 kinases’ activities, effective treatments may require multiple small molecule inhibitors targeting several TDP-43 kinases to fully prevent TDP-43 phosphorylation. Similarly, phosphatase activation could be used to drive clearance of phosphorylated TDP-43, although given the many known targets of calcineurin, PP1, and PP2, this strategy would need to be carefully evaluated to screen for potential adverse effects. Another strategy could be targeted clearance of phosphorylated TDP-43, potentially using an immunotherapy approach, or by enhancing endogenous cellular clearance mechanisms such as autophagy. Given the interplay among TDP-43 phosphorylation, aggregation, and cellular localization, treatments focused on disaggregation or restoration of nuclear localization could reduce TDP-43 phosphorylation as a downstream consequence, with potential neuroprotective benefits. There is an urgent need for effective therapies for TDP-43 proteinopathies; modifying TDP-43 phosphorylation holds both promise and challenge for ALS and FTLD-TDP.


In this study, a basic design of area-arrayed graphene nanoribbon (GNR) strain sensors was proposed to realize the next generation of strain sensors. To fabricate the area-arrayed GNRs, a top-down approach was employed, in which GNRs were cut out from a large graphene sheet using an electron beam lithography technique. GNRs with widths of 400 nm, 300 nm, 200 nm, and 50 nm were fabricated, and their current-voltage characteristics were evaluated. The current values of GNRs with widths of 200 nm and above increased linearly with increasing applied voltage, indicating that these GNRs were metallic conductors and a good ohmic junction was formed between graphene and the electrode. There were two types of GNRs with a width of 50 nm, one with a linear current–voltage relationship and the other with a nonlinear one. We evaluated the strain sensitivity of the 50 nm GNR exhibiting metallic conduction by applying a four-point bending test, and found that the gauge factor of this GNR was about 50. Thus, GNRs with a width of about 50 nm can be used to realize a highly sensitive strain sensor. In an aging society with a declining birthrate, the development of structural health-monitoring systems is extremely important in order to maintain a secure and reliable society for human beings through early detection of material damage. Especially in basic infrastructures such as energy plants and transportation systems, online damage-monitoring technology is strongly required due to the complexity of the systems and the shortage of skilled engineers due to the declining birthrate and aging population. In order to quantitatively evaluate material damage and prevent various failures, it is necessary to monitor the stress and strain fields of components in operation with high sensitivity and low cost; in other words, there is a strong demand for highly sensitive strain sensors. For these needs, strain sensors based on carbon nanoparticles, such as carbon nanotubes and graphene, have attracted attention as a viable alternative to conventional sensors based on metallic or semiconducting materials, mainly due to their superior electrical properties. Various sensors based on carbon nanomaterials have been proposed and practically used, and most of them are made by adding carbon fillers to a polymer matrix and forming an electrical network between the carbon fillers to provide high piezoresistivity ,2,3,4,5.5 cm2 V−1 s−1 [Recently, much attention has been focused on graphene as an available material for strain sensors with high sensitivity and large deformability . Graphen V−1 s−1 ,8,9,10.  V−1 s−1 . ZGNRs f V−1 s−1 . On the  V−1 s−1 ,14,15,16In this paper, we propose a basic design of an area-array-type, GNR-based strain sensor that can measure micro-strains with high sensitivity and high resolution. Each GNR arranged in an area array works as a strain gauge, and thus the distribution of strain in the area to be measured can be evaluated. In order to realize a GNR-based high-sensitivity strain sensor, it is essential to fabricate a high-quality GNR stably. High-quality GNRs can be fabricated by two methods: the bottom-up method and the top-down method. The bottom-up method is a direct synthesis of GNRs using synthetic chemical methods. The remarkable advantage of the bottom-up approach is the accuracy in controlling the ribbon width, edge structure, and film thickness when forming thin films at the atomic level, enabling accurate GNR structures with angstrom-level resolution and obtaining physically smooth and chemically clean edges ,18,19. HIn order to establish a stable and reliable fabrication process of the device using GNRs, the synthesis of a high-quality, single-layer graphene sheet is one of the important factors. In this study, graphene was grown on the inner surface of a Cu foil pocket structure by applying thermal LPCVD  using acetylene . Since t3. After washing the etched sample with dilute HCl and pure water, the cleaned sample was placed on a silicon substrate with a 300 nm thick thermal oxide film, and the PMMA layer was removed with acetone. An example of the transferred graphene is shown in In order to fabricate a GNR structure, the graphene synthesized on the Cu foil was transferred onto an oxidized silicon substrate. In this study, a wet transfer process was applied using PMMA (polymethyl methacrylate). This transfer process is also known as the PMMA-assisted transfer process . At firs2 plasma. The etching rate of PMMA with O2 plasma was 30 to 40 times larger than that of HSQ, and thus the HSQ pattern could be transferred to the graphene layer [The patterning process using EB lithography is described as follows. After the graphene was transferred onto the Si substrate, a Pt layer with a 5 nm thickness and a Au layer with an 80 nm thickness were deposited continuously by EB deposition using a stencil mask to form metal electrodes. After deposition of the metal electrodes, GNRs were patterned between the electrodes using the EB lithography technique. First, PMMA was coated and cured on graphene, and then HSQ (hydrogen silsesquioxane: XR1541-006 Toray Dow Corning) was spin-coated as a negative resist to form an HSQ/PMMA laminated resist layer. As PMMA has a high affinity for graphene, sufficient adhesion can be achieved with low-temperature curing compared to the case of a single layer of HSQ. Next, line patterns ranging from 50 nm to 400 nm in width and 18 μm in length were irradiated onto HSQ and developed with TMAH (tetramethylammonium hydroxide) to form the patterns. During the development, the PMMA layer was not dissolved in TMAH, and only HSQ was developed. Finally, PMMA was removed by RIE with One layer . An exam−1 and corresponds to carbon–carbon bonds; the second peak is called the D band, which appears at 1350 cm−1 and indicates the presence of disordered carbon atoms, or defects. The last peak, called the 2D band, appears at 2700 cm−1 and is related to the total number of layers in the graphene sheet. The relative intensity of the D and G bands (ID/IG) corresponds to the defect density in the observed region, and the relative ratio of I2D to IG corresponds to the number of layers in the graphene sheet. Therefore, the quality of graphene sheets improves with decreasing ID/IG values, and if the I2D/IG value is greater than 2.0, the graphene is considered to be composed of a single layer. For the fabricated GNRs, Raman spectroscopy was used to verify whether graphene existed under the resist layer and whether the etching removed the unused graphene. As shown in 2D/IG ratio was approximately 2.6, which indicated the presence of single layer of graphene. On the other hand, the intensity of the D band increased compared to graphene before the GNR fabrication process, with the ID/IG ratio increasing from 0.09 [The quality of the fabricated GNRs was evaluated by Raman spectroscopy. When a laser beam is irradiated on the surface of graphene, three typical Raman shifts are known to appear in the resulting reflection spectrum. One is called the G band, which appears at 1590 cmrom 0.09  to 0.4. D/IG < 0.05 sold by BGT Material Limited is 300–600 Ω/square [Ω/square . The sheSince it has been theoretically shown that AGNRs exhibit piezoresistive effects regardless of whether their initial electrical conduction properties are metallic or semiconducting , it was The strain dependence of the measured resistance of GNR is shown in In this study, we fabricated area-arrayed GNRs and evaluated the quality and strain dependence of their electrical conduction properties in order to realize a GNR-based strain sensor applicable in structural health monitoring. GNRs with widths ranging from 400 to 50 nm were fabricated by EB lithography between metal electrodes with 18 μm spacing. The I–V characteristics were evaluated for the fabricated GNRs. The current values of GNRs with widths of 400, 300, and 200 nm increased linearly with increasing applied voltage. It was confirmed that fabricated GNRs with a width of 200 nm or more exhibited metallic properties, and good ohmic contact was obtained between graphene and metal electrodes. The sheet resistance of GNRs was also evaluated based on the measurement results. The sheet resistances of GNRs with widths of 400 and 300 nm were 510–750 Ω/square, and the sheet resistance of GNRs with width of 200 nm was about 1000 Ω/square. The sheet resistance of GNRs with a width of 200 nm increased, but the GNR fabrication process developed in this study did not cause significant damage. On the other hand, for GNRs with a width of 50 nm, there were two types of GNRs: one showing metallic conduction in which the current value increased linearly with increasing applied voltage, and the other in which the current value increased nonlinearly. Theoretically, GNRs with ribbon widths of 100 nm or less are known to exhibit piezoresistive effects. Therefore, GNRs with a width of 50 nm were subjected to strain in a four-point bending test, and the strain dependence of electrical resistance was evaluated. As a result, a maximum gauge factor of about 50 was obtained in a strain range of 0.08% for GNRs showing metallic conductivity. This result indicated that GNRs could be applied to the development of highly sensitive strain sensors. Thus, we have successfully fabricated low-damage GNRs and demonstrated the possibility of developing highly sensitive strain sensors by applying GNRs.


Combining graphene with other nanomaterials can further tune the properties of graphene, and also can enhance the characteristics of the hybrid materials produced by the addition of this monoatomic-thick carbon layer. The range of possible applications of such hybrid materials is quite vast, including but not limited to energy storage, sensing, biomedicine, photocatalysis, or optoelectronics. This Special Issue presents several of the latest advances in the synthesis and characterization of graphene-based hybrid materials and nanostructures, with relevance for the development of sodium-ion batteries , NO2 sen sensors , or the  sensors . The fab sensors .0.2Ni0.25Mn0.75Oδ to form a sodium-ion cell. The so-obtained SIB was cycled at various rates and its performance was evaluated by monitoring the capacity as a function of the discharge rate and cycle number. It was confirmed that the battery was able to reversibly cycle sodium ions. The cell’s power response was good, and the cell cycled for over 100 cycles with capacities ranging from 120 up to 160 mA·h·g−1.In recent years, several intercalation materials have been investigated as potential electrodes for sodium-ion batteries (SIB). In the paper by Prosini et al. , the aut2 at room and elevated temperatures. It was shown that the FG particle fraction affects the relative response via the efficiency of charge transfer between the NO2 molecules and FG layers. The authors demonstrated that highly fluorinated graphite films provided a greater increase in the sensor response as compared to less fluorinated films.Graphene-based materials have been widely studied as sensitive layers of resistive gas sensors, as the electrical conductivity of graphene is strongly sensitive to surface adsorbates. Significant efforts were conducted to improve the response and recovery time of these sensors by controlling the functionalization of graphene. Sysoev et al.  reported−2, while higher fluences yielded web-like surface structures. The sheet resistance and the wettability of both pristine and graphene-doped PS were significantly affected by the applied laser treatment. The construction of different LIPSS structures is very important, for example, in studies involving cell-substrate interaction, cell guidance, or tailoring protein adsorption on biomimetic surfaces.Fajstavr et al.  reported2S on its surface. Preserving the high LSPR intensity of silver nanoparticles is of key importance in potential applications. Our group [Sensors based on the localized surface plasmon resonance (LSPR) of silver or gold nanoparticles are sensitive analytical tools which can be applied in chemical and biosensing. However, silver has poor stability under ambient conditions, forming Agur group  reportedIn a review by Barshutina et al. , researc


We compare the polarizability valuecomputed using density functional theory with the output from previouslypublished experimental data on the electrostatically controlled interlayerasymmetry potential in BLG and data on the on-layer density distributionin tBLG. We show that monolayers in tBLG are described well by polarizabilityα The manifestations of the qualitative change ofelectronic characteristics of both Bernal (BLG) and twisted (tBLG)bilayer graphene, produced by electrostatic gating3 and interlayer misalignment,5 were observedin numerous experimental studies of the electronic transport in graphene-basedfield-effect transistor (FET) devices. These versatile electronicproperties make FETs based on BLG and tBLG an attractive hardwareplatform for applications tailored8 for various quantum technologies.While, over the recent years, the fundamental electronic propertiesof bilayer graphene have been intensively studied, a mundane but practicalcharacteristic of this material related to the out-of-plane dielectricsusceptibility of graphene layers largely escaped attention of thoseinvestigations, despite several already recorded indications13 of its relevance for the quantitative modeling of the operationof graphene-based FET devices.Bilayer grapheneab initio density functional theory (DFT). We use that toestimate the effective dielectric susceptibility, ϵz, of graphene and to design a recipe for implementingit in the self-consistent description of electrostatics in bilayers,both twisted and with Bernal stacking. For tBLG with twist anglesoutside the magic angle range,8 we perform a mesoscaleanalysis of the on-layer carrier densities, finding a good agreementwith the earlier observations.13 Then, we implement the same recipe in the analysis of the interlayerLandau level pinning in strongly twisted bilayers. In this case, wealso find an excellent agreement between the theoretical results andthe measurements performed on a newly fabricated FET with a 30°-twistedtBLG. Finally, we take into account the out-of-plane dielectric susceptibilityof a single graphene layer in the self-consistent analysis of theinterlayer asymmetry gap in Bernal bilayer graphene,1 improving on the earlier calculations15 and successfully comparing the computedgap dependence on the vertical displacement field, Δ(D), with the earlier-measured interlayer exciton energiesin gapped BLG.16The out-of-plane dielectric susceptibilityof a single graphenelayer stems from the polarizability of its carbon orbitals, that is,from the mixing of π and σ bands by an electric fieldoriented perpendicular to the 2D crystal. Hence, we start by computingthe out-of-plane polarizability of a graphene monolayer using 17 with ultrasoft pseudopotentials.We use a 53 × 53 × 1 k-point grid, a largeplane-wave cutoff of 566 eV, and a variety of interlayer distances c along the z-axis to compute the totalenergy, Dz/ϵ0, centered on the carbon sites ofthe graphene layer (D being the displacement fieldand −c/2 < z < c/2). Then we determine (c using the relation δα(c) ∝ c–1, we fit the obtainedDFT data with α(c) = α∞ + a/c + b/c2 and find αDFT ≡ α(c → ∞) = 11Å3 per unit cell of graphene with the Perdew–Burke–Ernzerhof(PBE) functional and αDFT = 10.8Å3 with the local density approximation (LDA).To determine thetheoretical value of the out-of-plane dielectricpolarizability of a graphene monolayer, we employ the CASTEP plane-wave-basisDFT codeetermine  the pola3 = 10.9 Å3, and when recalculated into an effective “electronic thickness” 20 We also compared thecomputed DFT values with the polarizability computed using the variational(VMC) and diffusion (DMC) quantum Monte Carlo methods26 implemented in the CASINO code.27 Inthese calculations, we used the DFT-PBE orbitals generated using theCASTEP plane-wave DFT code28 and the orbitalsbeing rerepresented in a localized B-spline “blip” basis.The localized basis improves the scaling of the quantum Monte Carlo(QMC) calculations and allows the use of aperiodic boundary conditionsin the z-direction. The Jastrow correlation factorcontained isotropic electron–electron, electron–nucleus,and electron–electron–nucleus terms as well as 2D plane-waveelectron–electron terms,29 all optimizedusing VMC energy minimization.30 The DMCpart of the calculations was executed with a time step of 0.01 Ha–1 and a target population of 4096 walkers. The resultingQMC out-of-plane polarizability of graphene is α ≈ 10.5± 0.2 Å3, which is also close to the above-quotedDFT-LDA value, so that, in the analysis below, we will use αDFT = 10.8 Å3 for the polarizabilityof the graphene monolayer.These DFT values are close to the DFT-PBE polarizability reportedin ref , α= 0.8DFT todescribe the on-layer potentials andcharges in bilayers, as a function of doping and vertical displacementfield, D. For this, we note that z-polarization of carbon orbitals in each monolayer is decoupled fromthe charges hosted by its own π-bands because of mirror-symmetriccharge and field distributions produced by the latter, see in u, in the top and bottom layers of a bilayer, each with the electrondensity nb/t, has the formz is the effective out-of-plane dielectricsusceptibility, and d is the distance between thecarbon planes in the bilayer.For the analysis below, we use d = 3.35 Å, resultingin ϵz = 2.6 for BLG, and d = 3.44 Å (as in turbostratic graphite31), leading to ϵz ≈ 2.5 for tBLG. This expression is applicable to the descriptionof both BLG and tBLG in a FET, improving on the earlier-publishedstudies15 where theout-of-plane dielectric susceptibility of graphene layers was missedout in the self-consistent band structure analysis.Now, we will use the microscopicallycomputed polarizability α5v =10.2 · 106 m/s is Dirac velocity in monolayer graphene. 4 characteristic low-energy bands, illustrated in 1/ℏvK ≡2° . This spectrumfeatures two Dirac minivalleys at κ and κ′  multiterminaltBLG FET shown in the inset in T = 2 K) tBLG resistivity at zero (B = 0) and quantizing magnetic field. In the experimentally studieddevice, tBLG was encapsulated between hBN films on the top and bottom,thus providing both a precise electrostatic control of tBLG for B = 0 measurements and its high mobility, enabling us toobserve the quantum Hall effect at a magnetic field as low as B = 2 T. The measured displacement field and densitydependence of resistivity is shown in the form of color maps on theright-hand side panels in B = 0 and B = 2 T, respectively, where the form of “bright spots”of Rxx that appear ineach of these two cases is affected by the interlayer charge transfer,controlled by tBLG electrostatics in In fact, the weakest interlayerhybridization, λ →0, appears in “maximally” misaligned layers in a tBLGwith θ = 30°. In that case, the comparison between thetheory an experiment is simplified by that B = 0, we assumed elasticscattering of carriers from residual Coulomb impurities in the encapsulatingenvironment with a dielectric constant ϵ (ϵ ≈ 5for hBN), with an areal density nc, screened jointly by the carriers in the top and bottom layers.The screening determines32 the Fourierform factor of the scatterers3232 the density of states, γt/b, cancels out from each xx = ρtρb/[ρt +ρb], dependent on the carrier densityonly through the wavenumber transfer, 2kFt/b sin(φ/2), and screening.This produces ridge-like resistance maxima at kFt = 0 or kFb = 0, that is, whenFor a quantitative comparison of the measured andmodeled tBLGtransport characteristics at Lines corresponding to the above relationare laid over the experimentallymeasured resistivity map for a direct comparison.Nt and Nb, to each other and to their commonchemical potential, μ. As a result, we find that B = 2 T and Γ ≈ 0.5 meV is mapped in tot = hn/eB (n = nt + nb). Here, the “bright”high-DoS spots indicate the interlayer LL pinning conditions, whereasthe “dark” low-DoS streaks mark conditions for incompressiblestates in a tBLG. We compare this DoS map with ρxx measured in the quantum Hall effect regime (similar to theones observed earlier10 in other tBLG devices), wherethe high values of Rxx manifest mutual pinning of partially filled LLs,and the minima correspond to the incompressible states. To mention,the computed pattern broadly varies upon changing ϵz, whereas the value of ϵz = 2.5 gives an excellent match between the computed and measuredmaps in For comparisonbetween the theory and experiment at quantizingmagnetic fields, we studied the Landau level pinning between the twographene monolayers. In a magnetic field, graphene spectrum splitsinto Landau levels (LLs) with energies 1 in BLG, taking into account out-of-planepolarizability of its constituent monolayers. In this case, we use DFT using d = 3.35 Å,and the BLG Hamiltonian1ξ ≡ ξkx + iky, k = is the electron wave vector in thevalleys Kξ = ξ, ξ = ±. The computed sublattice amplitudes, Finally, we analyze the electrostaticallycontrolled asymmetrygapu, anda band gap, Δ, in the BLG spectrum  largely overestimate their values. At the sametime, the values of u and Δ obtained usingϵz = 2.6 appear to be less thanthe exciton energy measured in optics, for interlayer coupling acrossthe whole range 0.35  < γ1 <  0.38 eVcovered in the previous literature.42 This discrepancy may be related to that the interaction terms inthe electron self-energy are only partially canceled by the excitonbinding energy.37 It may also signal thatthe out-of-plane monolayer polarizability, α, is reduced by∼10% when it is part of BLG, as the values of Δ computedwith ϵz = 2.35 and γ1 = 0.35 eV agree very well with the measured optical gap values.The on-layer potentialenergy difference,  in refs , 14), a, au, and in refs ) and wit3, accounts verywell for all details of the electrostatics of twisted bilayers, includingthe on-layer electron density distribution at zero magnetic fieldand the interlayer Landau level pinning at quantizing magnetic fields.For practical applications in modeling of FET devices based on twistedbilayers, the polarizability of monolayer graphene can be convertedto its effective dielectric susceptibility, ϵz ≈ 2.5, which should be used for the self-consistentelectrostatic analysis of tBLG using In summary, the reported analysisof the out-of-plane dielectricsusceptibility of monolayer graphene shows that the latter plays animportant role in determining the electrostatics of both Bernal andtwisted bilayer graphene. We found that the DFT-computed polarizabilityof the monolayer, α = 10.8 Å