Fast, cost effective, single-shot DNA sequencing could be the prelude of a new era in genetics. As DNA encodes the information for the production of proteins in all known living beings on Earth, determining the nucleobase sequences is the first and necessary step in that direction. Graphene-based nanopore devices hold great promise for next-generation DNA sequencing. In this work, we develop a novel approach for sequencing DNA using bilayer graphene to read the interlayer conductance through the layers in the presence of target nucleobases. Classical molecular dynamics simulations of DNA translocation through the pore were performed to trace the nucleobase trajectories and evaluate the interaction between the nucleobases and the nanopore. This interaction stabilizes the bases in different orientations, resulting in smaller fluctuations of the nucleobases inside the pore. We assessed the performance of a bilayer graphene nanopore setup for the purpose of DNA sequencing by employing density functional theory and non-equilibrium Green’s function method to investigate the interlayer conductance of nucleobases coupling simultaneously to the top and bottom graphene layers. The obtained conductance is significantly affected by the presence of DNA in the bilayer graphene nanopore, allowing us to analyze DNA sequences. Sequencing DNA is the first step on the route towards so-called personalized or precision medicine1367910121467812In either approach, graphene has been heralded as an ideal candidate for a one-atom-thin membrane1920212324252628One possibility to simplify the fabrication process and simultaneously increase the transverse current is the use of a graphene nanopore to identify single nucleobases19303125et al.et al.et al.41In this work, we address these issues by investigating an alternative setup, namely a pair of aligned nanopores in bilayer graphene arranged in the Bernal (AB) stacking. The upper layer and bottom layer of graphene act as two separate electrodes which are connected by nucleobases traveling through the nanopores see . The por35As the DNA passes through the pore, there are many possibilities of molecular orientations with respect to the nanopore. We simulated DNA sequencing in a prototypical graphene device using classical molecular dynamics simulations of DNA translocation through the pore under the effect of the environment. In this manner, we are able to analyse the nucleobase trajectories and the interaction between the nucleobases and the nanopore. We further investigated the electrical transport properties of the four nucleobases: guanine (G), adenine (A), cytosine (C), and thymine (T), using a bilayer graphene nanopore for the purpose of electrical contact and nucleobase identification. This was achieved using a combination of classical molecular dynamics simulations, state-of-the-art density functional theory (DFT), and non-equilibrium Green’s functions (NEGF). As the nucleobase from a single-stranded DNA molecule is placed in the bilayer graphene nanopore, the local interlayer conductance is modulated by the nucleotide bridging the two graphene layers, revealing the DNA sequence.From the classical molecular dynamics (MD) trajectories one can analyse the interaction of the nucleobases with the nanopore. In θ1 formed between the normal to the aromatic rings in the nucleobase and the normal to the plane of graphene, and the angle between the projection of the normal to the aromatic rings on the plane of graphene and the vector lying on that same plane, which is normal to the circumference of the nanopore, θ2. We also analyzed the angle The hydrogen bonds are taken into account implicitly by the electrostatic and van der Waals terms of the particular classical force field being used. For the graphene pore edge, parameters for the phenol were taken, in order to determine the charge of the oxygen and hydrogen atoms, as well as the carbon adjacent to the oxygens. The absolute value of the classical partial charge is significantly higher in oxygen, compared to hydrogen, and so, the edge will be a strong hydrogen bond acceptor. This means that adenine and guanine should bind to the edge more strongly, because not only there are more hydrogen donor groups, but also they exhibit the correct spatial arrangement to keep the contact with the two graphene sheets at the same time. Although there are other hydrogen bond donor and acceptor groups available in each nucleobase, not all of them make hydrogen bonds with the pore, at the same time. For the purpose of counting the number of hydrogen bond donors, each hydrogen atom bound to a nitrogen/oxygen atom in a nucleobase is regarded a donor, so the amine groups of the nucleobases donate two hydrogen bonds. The orientation of the A and G nucleobases are closely transversal, with respect to the graphene plane, for G and A bases, because there are two effective hydrogen bond donors in each base, relatively spaced in the molecule, spanning almost all the length of the nucleobase plane, providing an efficient locking of the base position, across the two pores. Particularly for A, there is a potential effective hydrogen bond acceptor, which is the unprotonated nitrogen atom of the base, close to the amine group, which only makes effective contact with the pore if the base is tilted, with respect to the graphene plane. In the C nucleobase, there are also two effective hydrogen bond donors, but located in the same position, in a nitrogen atom, attached to the end of the nucleobase ring. In the T nucleobase, there is only one hydrogen bond donor. Therefore, in both cases the coupling with the graphene pore between the two pores happen only by that atom, and is significantly weaker, causing the disruption of the pore-nucleobase interaction along the MD simulation.Using the MD simulations, we can trace the nucleobase trajectory as the DNA is translocated through the bilayer graphene nanopore. From simulations for isolated single strands of DNA being pulled, we found that the distance between nucleobases is of the order of 5–7 Å, and thus larger than those observed in a relaxed structure or in double-stranded DNA. At these distances we do not expect significant interference in the electronic transport from bases above or below the base inside the pore. In that sense the system can be regarded to posses single-nucleobase resolution. Snapshots which provide the possibility of strong coupling strength between the nucleobase and nanopore were selected 6, poly(dG)6 and poly(dT)6. The bilayer graphene sheet was built in the AB Bernal stacking configuration. The edge of the 2-nm diameter nanopore was passivated by OH-groups.A classical molecular dynamics simulation was employed in order to obtain trajectories for the subsequent transport calculations. For this purpose, we set up the system as a DNA homopolymer crossing a pore of a bilayer graphene sheet, surrounded by water and counterions. For the DNA, 4 periodic single strand homopolymers were constructed, each of which containing 6 repetitions of the same base type, namely: poly(dA)σ = 3.50000e-01 and ε = 6.00000e-03). In this 4-layer system, a pore of diameter 2 nm was drilled in the center, and the graphene carbon atoms in the pore were saturated using hydroxyl functional groups. The graphene is periodic in x-direction , another layer of non-sticking material was used to cover the two sides of the graphene. A dummy atom was used for this purpose, with van der Waals parameters designed to mimic the presence of a protective coating layer  techniques based on DFTThe DFT simulation was performed with SIESTAHow to cite this article: Prasongkit, J. et al. Theoretical assessment of feasibility to sequence DNA through interlayer electronic tunneling transport at aligned nanopores in bilayer graphene. Sci. Rep.5, 17560; doi: 10.1038/srep17560 (2015).


This exam consists of 235 multiple-choice questions with five options, some of which include images provided in a separate booklet. The aim of this study was to analyze the technical quality of the multiple-choice questions included in the MIR exam over the last five years.The main factor that determines the selection of a medical specialty in Spain after obtaining a medical degree is the MIR  guidelines. We looked for 18 different issues included in the manual, grouped into two categories: issues related to testwiseness and issues related to irrelevant difficulties.The final number of questions analyzed was 1,143. The percentage of items based on clinical vignettes increased from 50 % in 2009 to 56-58 % in the following years (2010–2013). The percentage of items based on an image increased progressively from 10 % in 2009 to 15 % in 2012 and 2013.The percentage of items with at least one technical flaw varied between 68 and 72 %. We observed a decrease in the percentage of items with flaws related to testwiseness, from 30 % in 2009 to 20 % in 2012 and 2013. While most of these issues decreased dramatically or even disappeared , the presence of non-plausible options remained frequent.With regard to technical flaws related to irrelevant difficulties, no improvement was observed; this is especially true with respect to negative stem questions and “hinged” questions.The formal quality of the MIR exam items has improved over the last five years with regard to testwiseness. A more detailed revision of the items submitted, checking systematically for the presence of technical flaws, could improve the validity and discriminatory power of the exam, without increasing its difficulty. Medico interno residente”, internal medical resident). The Ministry of Health has designed and organized this exam since 1978. The exam is officially defined as “a nationwide test in which the applicants will receive a total individual score, obtained from the sum of the result of a multiple-choice test, (carried out in the simultaneously established exam rooms in assigned locations in different regions of Spain), and the score derived from their academic merits” [The examination that regulates access to medical specialties in Spain is known as the MIR exam . At the beginning of the study, the five researchers got together to analyze a series of items in order to agree on common criteria for assessing the different issues. In addition, several meetings were held to discuss doubts that had arisen, resolving the issues by consensus among the five researchers.We adapted the NBME guidelines for the analysis . These gThe different issues included in the manual were grouped into the 18 categories listed in Table The data were entered into a Microsoft Excel 2010 Spreadsheet  and exported to STATA 12 for the analysis. We carried out a descriptive and inferential analysis. We use chi-squared tests to compare differences between years  and logistic regression to study tendencies throughout the five years, in the remaining analyses.This study did not involve any personal human data.The final number of questions analyzed was 1,143. A total of 32 questions were not included in the analysis as they had been discarded by the qualifying committee after the refutations from the examinees.The percentage of items based on clinical vignettes increased from 50 % in 2009 to 56-58 % in the following years (2010–2013). The percentage of items based on an image increased progressively from 10 % in 2009 to 15 % in 2012 and 2013. The interannual differences in the percentage of vignettes and images were not significant.There was an overall decrease in the percentage of issues related to testwiseness throughout the five years. The main reduction (31.6 % decrease) was observed between 2010 and 2011, with stable percentages of approximately 20 % in the last years  that disappeared in 2010 and remained adequate in the following years .With regard to logical clues Fig. , there ip < 0.001).In Fig. p < 0.001).Lack of uniformity in the options was a frequent flaw in the items studied. A highly significant improvement throughout the five years that were analyzed (from 10 to 4 %) is observed in Fig. p = 0.08) towards an increase in the percentage of flawed items is present.As shown in Fig. Figure Figure With regard to negative stems (including “except” or “not” in the lead-in), there is no improvement in the percentage of flawed items; in fact, there is actually a slight increase Fig. .p value of 0.03 .We also looked for writing/orthographic errors and outdated terms in the items; although the percentage was quite low, there were no significant differences throughout the years  did not vary significantly in the last five years (28 to 32 %).We carried out an additional analysis comparing the percentage of flawed items in questions including and not including clinical vignettes. The results showed no differences between them (the curves overlapped), except for answers “hinged” to the answer of a previous question, which were more frequent in questions with clinical vignettes. This difference was an expected finding, as most items based on an image also include a clinical vignette, and every image always has two related questions.Our study has found a high percentage of items with technical flaws in the MIR exams in the period 2009–2013. However, most of these flaws were related to irrelevant difficulties and the majority of the flaws related to testwiseness did indeed improve over the five-year period (with some of them even disappearing). The percentage of items with any type of technical flaw did not vary significantly over the last five years 68 to 72 %). According to similar studies in the literature: 28 to 75 % [8 to 72 %Technical flaws related to testwiseness favor the use of “tricks”, making it easier to answer the questions just by using test-taking abilities. Tarrant et al. observed that borderline students benefit from flawed items . From ouIrrelevant difficulties are those not associated with the subject that the question pretends to evaluate. They make the item more difficult, but they do not help to discriminate between students who are knowledgeable and those who are ignorant regarding the subject matter . A more Some subtypes of these technical flaws deserve more detailed analysis. In our study negative stems, and those that cannot be answered without looking at all the options (unfocused stem) were the most frequent mistakes; these same types of mistakes were also frequently found in other studies , 7. ItemPercentage of questions based on a clinical vignette or on an image increased over the five-year period studied. As commented in the results section, the percentage of item flaws did not increase in the questions with a clinical vignette (with the exception of “hinged” answers). Thus, the inclusion of clinical vignettes does not decrease the technical quality of the exam. On the other hand, previous studies have indicated that clinical vignettes and images add some practical components to the knowledge evaluated in a MCQ exam, including differential diagnosis and integration of knowledge from different areas , 14. TakColor images used in the exam are provided in a separate booklet. Typically, image-associated items are presented in pairs (two questions per image); this is probably done for economic reasons, as color printing is expensive. Our study shows that of the 10-15 % of items that are associated to an image, in nearly half of them (4-7 %), the answer is “hinged” to the answer of another item . This finding implies that “hinging”  or “cueing” (one of the questions provides clues to answer the other one) was present in most of the two-item image-related clusters. “Cueing” makes answering a question much easier for students with expertise in test-solving , 15. “HiA limitation of our work is the lack of individual performance data analysis to complement the technical flaw study. Modern validity theory is a unitary concept that goes beyond the technical quality of the issues . This adWe agree with Palmer et al.  that a wThe technical quality of MIR exam items has improved throughout the last five years, especially with regard to flaws related to testwiseness, but there is still quite a bit of room for improvement. In this regard, Medical Education Units may have a fundamental role in the training of evaluators. We suggest three actions that could help improve the future quality of the MIR exam: 1) Instruct the professionals involved in writing and selecting MIR questions regarding how to avoid and detect technical flaws; 2) increase the number of questions submitted in order to have a larger pool for the selection process; 3) insist on the most frequent flaws: negative stems, items that cannot be answered without reading the options, hinged questions, and non-plausible distractors. Indeed, the ministry of Health has recently announced that the number of options will be reduced from 5 to 4 in the next MIR exam in 2016.Some of the conclusions from our study may also be relevant beyond the MIR exam. Our results suggest that testwiseness issues are easier to eliminate than irrelevancy ones. Also, they indicate that clinical vignettes, which have many advantages in terms of evaluation, do not have a higher proportion of technical flaws. However, the use of two questions per image has a high risk of hanging or cueing flaws.


C. elegans model of ALS. In this kinome-wide survey, we identified homologs of the tau tubulin kinases 1 and 2 (TTBK1 and TTBK2), which were also identified in a prior screen for kinase modifiers of TDP-43 behavioral phenotypes. Using refined methodology, we demonstrate TTBK1 and TTBK2 directly phosphorylate TDP-43 in vitro and promote TDP-43 phosphorylation in mammalian cultured cells. TTBK1/2 overexpression drives phosphorylation and relocalization of TDP-43 from the nucleus to cytoplasmic inclusions reminiscent of neuropathologic changes in disease states. Furthermore, protein levels of TTBK1 and TTBK2 are increased in frontal cortex of FTLD-TDP patients, and TTBK1 and TTBK2 co-localize with TDP-43 inclusions in ALS spinal cord. These kinases may represent attractive targets for therapeutic intervention for TDP-43 proteinopathies such as ALS and FTLD-TDP.Pathological aggregates of phosphorylated TDP-43 characterize amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), two devastating groups of neurodegenerative disease. Kinase hyperactivity may be a consistent feature of ALS and FTLD-TDP, as phosphorylated TDP-43 is not observed in the absence of neurodegeneration. By examining changes in TDP-43 phosphorylation state, we have identified kinases controlling TDP-43 phosphorylation in a  in vitro, and control TDP-43 phosphorylation in cellular and simple animal models of ALS. This has uncovered a molecular mechanism by which pathological phosphorylated TDP-43 can occur in disease. To determine whether changes in TTBK1/2 protein are contributing to TDP-43 pathology, we examined diseased brain and spinal cord tissue from patients with ALS or FTLD-TDP. We observed changes in the abundance of TTBK1 and TTBK2 in disease-affected neurons, and the coexistence of TTBK1/2 with phosphorylated TDP-43 aggregates in both FTLD-TDP and ALS. Therefore, increased abundance or activity of TTBK1 or TTBK2 may contribute to the neurodegeneration observed in ALS and FTLD-TDP.Aggregated proteins are a hallmark of many neurodegenerative diseases. In ALS and FTLD-TDP, these aggregates contain abnormal TDP-43 modified by phosphorylation. Protein phosphorylation normally controls protein activity, stability, or location, but in some neurodegenerative diseases the phosphorylated proteins accumulate in excess. Kinases are the enzymes responsible for protein phosphorylation. We have identified two TDP-43 kinases, TTBK1 and TTBK2, using a novel approach combining reverse genetics and biochemical screening to identify the kinases responsible for changes in TDP-43 phosphorylation. We show TTBK1 and TTBK2 directly phosphorylate TDP-43  TARDBP, were found to cause ALS in a subset of affected families, supporting a causal role for TDP-43 in disease initiation C. elegans, Drosophila, zebrafish, mice, and rats have demonstrated neurotoxicity resulting from mutant TDP-43 Ubiquitinated, hyperphosphorylated inclusions of the protein TDP-43 characterize disease-affected neurons in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD-TDP) TDP-43 undergoes a number of pathological modifications in disease-affected neurons including ubiquitination, phosphorylation, and proteolytic processing. These modifications may promote aggregation and the formation of detergent-insoluble inclusions. The precise molecular cause underlying neurotoxicity in most TDP-43 proteinopathies remains unclear, although the toxicity of mutant TDP-43 expressed in multiple model systems indicates it may be acting through a gain-of-function mechanism via aberrant interactions with proteins and/or nucleic acids C. elegans model of TDP-43 proteinopathy exhibiting TDP-43 phosphorylation dependent neurodegeneration and neurotoxicity; in C. elegans, phosphorylation of TDP-43 at serines 409 and 410 suffices to promote TDP-43 mediated neurotoxicity C. elegans, because detectable phosphorylated TDP-43 remains in the absence of CDC7. Inhibition of the kinases CDC7 or CK1 has also been shown to reduce but not eliminate TDP-43 phosphorylation in cultured cells C. elegans. We have identified homologs of the tau tubulin kinases TTBK1 and TTBK2 and characterized their function as regulators of TDP-43 phosphorylation. TTBK1/2 may be attractive drug targets for therapeutic interventions in TDP-43 proteinopathies such as FTLD-TDP and ALS.In order to investigate the causes driving pathological TDP-43 phosphorylation, we have developed a C. elegans. We have assembled an RNAi library targeting 451 predicted kinase genes in C. elegans . This library has been previously employed to identify kinase modifiers of TDP-43 dependent behavioral phenotypes, and identified CDC7 as a direct TDP-43 kinase responsible for promoting TDP-43 neurotoxicity C. elegans model as detectable phosphorylation at S409/410 is still observed in a cdc-7(−/−) null mutant background. Thus other kinases play conserved roles phosphorylating TDP-43, and previous behavior-based screening may have failed to uncover kinases with multiple roles in vivo, or kinases whose loss of function could adversely impact motor function or viability independent of TDP-43. To identify additional TDP-43 kinases, a direct biochemical assay of TDP-43 phosphorylation in TDP-43 transgenic C. elegans was used to screen for alterations in pS409/410 TDP-43 phosphorylation. Populations of transgenic C. elegans expressing ALS-mutant M337V TDP-43 were grown on bacteria producing double stranded RNA targeting each kinase, then harvested and tested by immunoblot for changes in TDP-43 phosphorylation  family members C. elegans suggests a diversification of functional roles for the TTBK1/2 like kinases in the nematode.To identify TDP-43 kinases, we undertook a comprehensive survey utilizing kinase-targeting RNAi coupled with direct immunoblot detection of changes in TDP-43 phosphorylation in rylation . Transgecdc-7(−/−), H05L14.1(−/−), and dkf-2(−/−), dramatically reduce TDP-43 phosphorylation with only moderate or no changes in total levels of TDP-43, consistent with the results from the initial RNAi screen (dkf-2(−/−) exhibited significant differences in total TDP-43 levels. cdc-7(−/−) has been previously characterized as a TDP-43 kinase dkf-2(−/−).RNAi can inactivate multiple genes simultaneously depending on their sequence similarity, potentially confounding the identification of any single gene responsible for TDP-43 phosphorylation. To unambiguously determine the effects of single kinase gene loss of function on TDP-43 phosphorylation, we generated TDP-43 transgenic animals with viable deletion mutants eliminating the kinase active domain of each candidate gene of interest . Each ofi screen . We obsei screen , but thecdc-7(−/−);TDP-43, H05L14.1(−/−);TDP-43 or dkf-2(−/−);TDP-43 had a more natural and vigorous movement profile relative to the TDP-43 transgene alone. We assessed motor function by measuring the average dispersal velocity of the animals, and found significant improvements compared to TDP-43  C. elegans and other ecdysozoa. The H05L14.1 kinase domain has 40% sequence identity to the highly homologous tau tubulin kinases TTBK1 and TTBK2 at the amino acid level   . PRKD2/3immunity  [29], [3activity . Our prein vitro  to decrease levels of TTBK1 gene expression in mammalian cultured cells. We have modeled pathological TDP-43 phosphorylation in the mouse motor neuron-like NSC-34 cell line using the chemical trigger ethacrynic acid (EA). EA acts by depleting cytosolic and mitochondrial glutathione, resulting in robust TDP-43 phosphorylation Both TTBK1 and TTBK2 are expressed in the brain, although TTBK2 is expressed in other tissues as well Of the six ALS cases examined, only two had phospho-TDP-43 aggregates in the frontal cortex and hippocampus, while all six demonstrated phospho-TDP-43 aggregates within spinal cord. ALS spinal cord motor neurons immunoreactive for phospho-TDP-43 pathology also co-labeled with TTBK1 and TTBK2 . Of the C. elegans demonstrated a causal relationship between neurodegeneration and S409/410 phosphorylation of TDP-43 C. elegans behavior-based screening tool to identify TDP-43 kinases C. elegans kinome by RNAi knockdown for modifiers of TDP-43 phosphorylation. For this survey we employed sensitive and specific S409/410 phosphorylation dependent TDP-43 antibodies C. elegans was conducted by testing deletion mutations within the kinase genes of interest. Three identified candidate kinases, cdc-7, dkf-2, and H05L14.1, reduced TDP-43 S409/410 phosphorylation and improved TDP-43 dependent behavioral phenotypes in C. elegans. TDP-43 is a known substrate of CDC7, as it was previously uncovered in a reverse genetic screen to identify modifiers of TDP-43 behavioral phenotypes dkf-2 and H05L14.1. Interestingly, H05L14.1 was a homolog of the mammalian tau tubulin kinases TTBK1 and TTBK2. Our previous behavior-based screen for TDP-43 kinases identified a different C. elegans homolog of TTBK1/2 as a TDP-43 kinase, although at the time we were unable to demonstrate a direct relationship between human TTBK1/2 and TDP-43 in vitro kinase reaction conditions, we demonstrate here that human TTBK1/2 are able to directly phosphorylate TDP-43. We then overexpressed TTBK1/2 in cultured human cells. TTBK1/2 overexpression in the absence of other stressors promoted robust phosphorylation of endogenous TDP-43. Furthermore, this phosphorylated TDP-43 localized to the cytoplasm in inclusion-like aggregates. We also found that reduction of TTBK1 mRNA levels attenuated TDP-43 phosphorylation in a chemically induced model of pathological phospho-TDP-43 accumulation. Finally, to explore whether TDP-43 kinase hyperactivity could underlie the etiology of TDP-43 proteinopathies, we immunostained tissue from FTLD-TDP and ALS for TTBK1/2. We observe increased TTBK1/2 in FTLD-TDP frontal cortex, and co-localization with TDP-43 positive aggregates in FTLD frontal cortex and ALS spinal cord. One possible explanation for these data is the observed differences in TTBK1/2 expression drives neurodegeneration in TDP-43 proteinopathies. Taken together these data support a pivotal role for TTBK1/2 hyperactivity in TDP-43 proteinopathy.Tandem phosphorylation at TDP-43 serines 409 and 410 (pS409/410) is a consistent and robust feature of TDP-43 pathology in ALS and FTLD-TDP. Our previous work in TDP-43 transgenic in vitro and in vivo. The kinases CK1 C. elegans, mutant TDP-43 still exhibits varying but detectable degrees of phosphorylation  in TTBK1 are associated with decreased Alzheimer's disease risk in studies of Spanish and Han Chinese populations Kinases regulating TDP-43 phosphorylation present an attractive target for therapeutic intervention in both ALS and FTLD-TDP. No specific small molecule inhibitors targeting TTBK1 or TTBK2 has been reported to date, despite their potential roles contributing to tauopathies by hyperphosphorylating tau. Development of brain penetrant TTBK1 and TTBK2 inhibitors may also provide a viable strategy for intervening in TDP-43 proteinopathy disorders including ALS and FTLD-TDP.C. elegans and maintained as previously described C. elegans were performed at room temperature unless otherwise noted. CK423 (TDP-43 M337V) and eri-1(mg366);lin-15b(n744);TDP-43 M337V transgenic strains were generated previously cdc-7(tm4391);TDP-43, CK602 H05L14.1(tm4720);TDP-43, CK600 dkf-2(tm4076);TDP-43, CK597 nsy-1(ok593);TDP-43, CK613 kin-20(ok505);TDP-43, CK574 mlk-1(ok2471);TDP-43, CK623 F39F10.3(tm4396);TDP-43.N2 (Bristol) was used as wild type C. elegans was derived from the C. elegans kinome project eri-1(−/−);lin-15(−/−) RNAi enhancing mutant background st generation gravid adults with 2nd generation L2–L3 animals were harvested by washing with M9 buffer into 96 well plates and frozen at −80°C, for subsequent immunoblot analysis. Each RNAi treated population was evaluated semi-quantitatively for reduction in phospho-TDP-43 relative to control treated animals. Positives candidates were retested for effects on TDP-43 phosphorylation by independent RNAi treatment and immunoblot, and the RNAi gene target for each plasmid was confirmed by sequencing.The list of predicted kinase genes in C. elegans β-tubulin levels were measured using monoclonal antibody E7 as a loading control as previously described Equivalent mixed-stage worm lysate fractions were loaded and resolved on precast 4–15% gradient SDS-PAGE gels and transferred to PVDF membrane as recommended by the manufacturer (Bio-Rad). On immunoblots, human TDP-43 was detected with a commercially available monoclonal antibody ab57105 (Abcam) directed against human TDP-43 amino acids 1–261. TDP-43 phosphorylated at S409/S410 was detected by a monoclonal antibody called anti phospho TDP-43 (pS409/410) available from Cosmobio . GST-TDP-43 (WT) and GST-TDP-43 (M337V) fusion proteins were purified from BL21 (DE3) expression host cells as previously described HEK 293 cells (ATCC) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% defined fetal bovine serum (FBS) and penicillin (50 IU/ml)–streptomycin (50 mg/ml). NSC-34 cells (Cedarlane Labs) and SHSY-5Y cells (ATCC) were cultured in DMEM/HAM's F12 (50/50) with 10% FBS and penicillin (50 IU/ml)–streptomycin (50 mg/ml).2+/Mg2+, then blocked in antibody buffer +10% normal goat serum. Primary antibody was applied and incubated for 1 hour at room temperature . Cells were washed 3×5 min in PBS/Ca2+/Mg2+, then re-blocked for 10 min. Appropriate secondary antibody was applied and incubated for 20 min at room temperature. Cells were again washed 3×5 min in PBS/Ca2+/Mg2+, counterstained with 300 nM DAPI and mounted with ProLong Gold antifade. Microscopy was performed on a Delta Vision microscope  using a 60× oil immersion objective, a sCMOS camera, and 2×2 binning. Image analysis was performed using softWoRx 6.0 Beta software.Cells were seeded onto poly-D-Lysine coated (Sigma Aldrich) 12 mm round glass cover slips in 24-well plates. Cells were transfected with the plasmid encoding TTBK2-GFP with GenePorter 2 (Genlantis) using the manufacturer's protocol. Cells were fixed for imaging in 4% formaldehyde 96 hours after transfection. Cells were washed 3×5 min in PBS/CaHEK 293 cells were treated with 150 µM ethacrynic acid (EA) for 5 hours to induce endogenous TDP-43 phosphorylation Transfection of plasmids containing full-length TTBK1 (pWO:TTBK1) and TTBK2 (TTBK2 GFP pFLAP dest) sequences RNA was purified from flash-frozen cell pellets using TRIzol Reagent (Life Technologies) according to the manufacturer's protocol. cDNA was made using iScript Reverse Transcription Supermix (Bio-Rad). qPCR was performed on an 7900HT Real Time PCR System (Applied Biosystems) using iTaq Universal SYBR Green Supermix (Bio-Rad).De-identified post-mortem brain tissue used in this study was determined to be an exempt from IRB review by the VA Puget Sound Health Care System Human Research Protection Program Director on December 29, 2011. Tissue used for these studies was obtained from the University of Washington Alzheimer's Disease Research Center brain bank , and the Indiana Alzheimer Disease Center brain bank, where consent for autopsy and permission for use of tissue in scientific experiments was obtained. FTLD and ALS cases were selected on the basis of having an autopsy-confirmed diagnosis of FTLD and FTLD-related disorders or ALS. Control samples were from de-identified neurologically healthy control participants, who were of a similar age.Primary antibodies used for immunohistochemistry were anti-TTBK1 , anti-TTBK2 , and anti-phospho TDP-43 409/410 ). In order to minimize variability, sections from all cases  were stained simultaneously for each antibody. Immunostained sections were analyzed using the computerized image analysis system, MicroComputer Imaging Device . Blinded assessment of optical density measurements were obtained relative to the proportional area for TTBK1 and TTBK2 immunostaining in frontal cortex grey matter (three separate readings per case). Data were averaged and are represented as mean +/− SEM. A two tailed Student's t-test was used to assess differences in TTBK1 and TTBK2 expression between cases and controls. For double label immunohistochemistry experiments, sections were first immunostained with anti-phospho TDP-43 and reaction product was visualized with nickel enhanced DAB (black). Sections were then immunostained with anti-TTBK1 or TTBK2 and visualized with DAB alone (brown). For double label immunofluorescence experiments, AlexaFluor 488 goat anti-rabbit and AlexaFluor 594 goat anti-mouse secondary antibodies (Molecular Probes) were used and autofluoresence was quenched with 0.1% Sudan Black S1 FigureC. elegans were harvested into 96-well plates prior to immunoblot analysis. Gene names and locations of kinases tested are presented in Table S1. Two rows from each plate were tested in alternating wells for each immunoblot. Labels above individual wells describe Row and Column information for each sample. Plate numbers are indicated at the left of the immunoblot. α-tubulin antibody is used as a load control. Candidates confirmed on repeat testing are boxed in blue.Immunoblot results from primary kinase RNAi screen. Populations of RNAi treated (PDF)Click here for additional data file.S2 FigureFull immunoblots of total TDP-43 levels. Full immunoblots from (PDF)Click here for additional data file.S3 Figuredkf-2 mammalian homologs identified by BLAST.  Cladogram of vertebrate homologs of the C. elegans proteins H05L14.1 and dkf-2. The entire C. elegans amino acid sequences of H05L14.1 or dkf-2 were compared against non-redundant reference sequences from the RefSeq protein database . (A) 2878 active hits were identified by BLAST with similarity to H05L14.1, and subsequently filtered to include only sequences from C. elegans or phylum chordata. The top 50 hits underwent multiple sequence alignment, alignment refinement, phylogenetic reconstruction, and are displayed in a cladogram, with branch support values in red dkf-2, filtered, and graphed as above. Homo sapiens GI# 5031689 is PRKD3. (C) H05L14.1 kinase domain has 40% identity to human TTBK1 and TTBK2. (D) dkf-2 has more than 70% identity to human PRKD2 and PRKD3. Sequence identity was calculated using Clustal W method for multiple sequence alignment. (E) Alignment report for H05L14.1, TTBK1, and TTBK2 kinase domain, including boxes around sequence that matches the consensus. (F) Alignment report for dkf-2, PRKD2, and PRKD3. Reports were generated using Lasergene MegAlign software for protein sequence analysis and alignment.H05L14.1 and (PDF)Click here for additional data file.S4 FigureIn vitro kinase assay controls. (A) Tau is a known substrate of TTBK2 E. coli were incubated with equivalent amounts of TTBK2 enzyme purified from cultured cells by two commercial suppliers . Phosphorylation was assessed by reactivity with AT270, a phospho-tau antibody recognizing tau phosphorylated at Thr181. (B) Purified TTBK1, TTBK2, and CDC7 can also phosphorylate TDP-43 at serines 403 and 404  in an in vitro kinase assay. (C) Histone H1 is a known substrate of PRKD2 (PDF)Click here for additional data file.S5 Figure(A) Phosphorylated TDP-43 is localized in a large discrete cytoplasmic aggregate following TTBK2 overexpression in HEK293 cells. (B) TTBK2 and phosphorylated TDP-43 co-localize in cells overexpressing TTBK2.(PDF)Click here for additional data file.S6 Figure(A) Treatment of HEK293 cells with a variety of cellular stressors failed to produce phosphorylated TDP-43. Bafilomycin and wortmannin are inhibitors of autophagy, PSI is a general proteasome inhibitor, cadmium chloride is a heavy metal, taxol is an inhibitor of microtubule dynamics, rotenone blocks the mitochondrial electron transport chain (creating reactive oxygen species (ROS)), pepstatin inhibits aspartic proteases, and paraquat catalyzes formation of ROS. (B) TTBK1 is reduced by nearly 80% following siRNA treatment in NSC-34 cells. Quantitative PCR measurements (qPCR) for TTBK1 mRNA levels are displayed in arbitrary units for an untreated control and cells treated with TTBK1 siRNA. (C) siRNA targeting TTBK1 reduce levels of TTBK1 protein, as detected by immunoblot. (D) TTBK1 protein levels are reduced by an average of 46%, following siRNA treatment in NSC-34 cells. Quantitation of TTBK1 protein levels from three independent experiments is graphed in arbitrary units of band intensity.(PDF)Click here for additional data file.S7 FigureAbcam, ab103944). (B) Antibodies tested for TTBK2 were TTBK2 Antibody N-term , Anti-Tau tubulin kinase 1 antibody , and TTBK2 Polyclonal Antibody . Antibodies underlined/bold above were used in further experiments and are boxed in red in the figure. (C–J) Peptide blocking experiments with the cognate immunizing peptide further demonstrates specificity of the selected TTBK1 and TTBK2 antibodies. Anti-TTBK1  (C–F) and anti-TTBK2  (G–J) were pre-incubated with a 50 fold excess of the blocking peptide  before proceeding with the immunostaining protocol and compared with immunostaining using antibody alone . ALS spinal cord  and FTLD frontal cortex . Scale bar = 100 um.Antibody Validation. (A) Studies examining expression of TTBK1 used commercially sourced antibodies including Anti-TTBK1 , Anti TTBK#1 , Anti-TTBK1 ((PDF)Click here for additional data file.S8 FigTTBK1/2 co-localize with phosphorylated TDP-43 in aggregates in ALS spinal cord. Double-label immunofluorescence of ALS spinal cord demonstrates additional neurons that significantly co-localize TTBK1 (upper panel) or TTBK2 (lower panel) with phospho-TDP-43 within neuronal cytoplasmic inclusions. Significance was determined using Pearson coefficient of colocalization.(PDF)Click here for additional data file.S1 TableC. elegans prior to testing by immunoblot . Control RNAi for each plate are highlighted in purple. L4440: empty vector RNAi control. unc-22: positive control for effective RNAi treatments, causing a strong paralyzed phenotype in treated TDP-43 worms. TDP-43: RNAi targeting the TDP-43 transgene is a positive control for suppression of TDP-43 phenotypes. Kinase RNAi treatments that caused C. elegans sterility or growth arrest, causing insufficient sample for protein detection by immunoblot, are highlighted in green. Kinase RNAi treatments that decreased TDP-43 phosphorylation in initial testing are highlighted in blue. Kinase RNAi treatments that reproducibly decreased TDP-43 phosphorylation in multiple independent experiments have names that are bolded and underlined.Kinase genes tested by immunoblot. 96-well plate locations of each RNAi treated population of (XLSX)Click here for additional data file.


Microfabricated electron emitters have been studied for half a century for their promising applications in vacuum electronics. However, tunable microfabricated electron emitters with fast temporal response and controllable electron emission still proves challenging. Here, we report the scaling down of thermionic emitters to the microscale using microfabrication technologies and a Joule-heated microscale graphene film as the filament. The emission current of the graphene micro-emitters exhibits a tunability of up to six orders by a modest gate voltage. A turn-on/off time of less than 1 μs is demonstrated for the graphene micro-emitters, indicating a switching speed about five orders of magnitude faster than their bulky counterparts. Importantly, emission performances of graphene micro-emitters are controllable and reproducible through engineering graphene dimensions by microfabrication technologies, which enables us to fabricate graphene micro-emitter arrays with uniform emission performances. Graphene micro-emitters offer an opportunity of realizing large-scale addressable micro-emitter arrays for vacuum electronics applications. Controlling the electron emission of microfabricated field emitters can be challenging. Here the authors report controllable and tunable graphene thermionic micro-emitters with well-defined turn-on voltages and switching times in the microsecond range and fabricate uniform micro-emitter arrays. An electron emitter for generating a free electron beam is the heart of vacuum electronic devices. Owing to the simplicity of fabrication and the ease of extracting large and stable emission current, traditional thermionic electron emitters remain prevail in most commercialized vacuum electronic devices. However, because of the bulky size and slow temporal response of thermionic electron emitters, vacuum electronic devices are usually in a large volume and have a slow switching speed, which have significantly limited their applications. In 1968, Spindt pioneered the fabrication of field electron micro-emitter arrays using microfabrication technologies3168101113Regarding the materials for the filaments of microfabricated thermionic emitters, it is highly desired from them to exhibit good electrical conductivity, high decomposition or melting temperature, proper work function and good chemical inertness, as learnt from the properties of the filaments of bulky thermionic emitters. More importantly, they have to be compatible with traditional microfabrication technologies and can be controllably tailored into microscale dimensions by microfabrication technologies. In recent years, two-dimensional layered materials have attracted great research interests because of their promise in field effect transistors1719212223172527In this paper, we report the miniaturization of thermionic emitters and the construction of the micro-emitter arrays by using microfabrication technologies and employing Joule-heated microscale graphene films as the filaments. Emission current of the graphene micro-emitters (GMEs) measured by a top electrode can be tuned by up to six orders of magnitude by a modest gate voltage of approximately 15 V, and a turn-on/off time of less than 1 μs is demonstrated for a GME. In contrast with microfabricated field emitters, whose turn-on voltage is difficult to control through microfabrication, the turn-on voltage of GMEs depends on graphene dimensions, enabling controllable and reproducible emission performances of GMEs by engineering graphene dimensions by microfabrication technologies. Consequently, micro-emitter arrays consisting of GMEs with uniform electron emission performances are successfully realized. The large-magnitude tunability by a modest gate voltage, fast temporal response and good controllability of emission performances make GMEs promising in realizing large-scale addressable micro-emitter arrays for vacuum electronics applications.2/Si wafer by microfabrication technologies. It consists of a microscale graphene film suspended between two Au/Cr electrodes and over a heavily doped Si bottom electrode. A scanning electron microscope (SEM) image of a GME is shown in 29Vb) and thus an electric current (Ib) are applied to the graphene emitter through the two Au/Cr electrodes to heat it up by Joule heating. To measure the emission current, a collecting voltage (Vc) is applied to a W electrode locating ∼200 μm above the graphene emitter. The heavily doped Si bottom electrode applied with a gate voltage (Vg) works as a gate electrode to tune the emission current measured by the upper W electrode. Ic) from a graphene emitter measured by the upper W electrode together with the current (Ig) measured by the bottom gate when Vb was ramped up from 2.0 to 3.5 V and Vc and Vg were fixed at 100 and 15 V, respectively. It can be seen that electron emission from the GME takes place at a turn-on voltage of only ∼2.8 V. When Vb is larger than the turn-on voltage, both Ic and Ig increase exponentially with Vb. The two currents increase synchronously with Ig being about two times larger than Ic, which is attributed to the larger collecting electric field (∼50 V μm−1) between graphene and the bottom gate than that between graphene and the top W electrode (∼0.5 V μm−1). Further experimental evidences to indicate that the measured electron emission is from graphene film are shown in Ic–Vg curves of the same GME as that in Vb increases from 3.10 to 3.35 V at 0.05 V intervals and Vc is fixed at 100 V. A Vc value of 100 V ensures that all those curves were measured at accelerating field regime without the retardation of space charge  of a SEM to detect the electrons emitted from a GME . Electro32Ic with Vb after being turned on . The low vacuum requirement for the operation of GMEs will significantly decrease the cost of GME-based vacuum electronic devices.In addition to the controllable and reproducible emission performances of GMEs, electron emission from a GME and a GME array can be driven by a voltage of as low as ∼2–3 V depending on graphene length  and 5. T3Vb is larger than 3.4 V in 323333Emission current of a GME is observed to exhibit a strong correlation with the bias voltage or internal electric field applied to it, rather than the applied Joule-heating power . An incrTo summarize, thermionic emitters are scaled down to the microscale for the first time by using microfabrication technologies and employing microscale Joule-heated graphene films as the filaments. Compared with traditional bulky thermionic emitters, GMEs possess the merits of microscale size, fast temporal response, large-magnitude local gate tunability by a modest gate voltage and being integratable by microfabrication technologies. Compared with microfabricated field emitters, GMEs exhibit controllable and reproducible electron emission performances, low working voltage and low requirement for operation vacuum. Possessing the advantages of both bulky thermionic emitters and microfabricated field emitters, GMEs open up a route of realizing large-scale addressable micro-emitter arrays for vacuum electronics applications. Because of the relative high work function of graphene and the high dissipation rate of Joule-heating power, emission efficiency and emission current density of GMEs are still overshadowed compared with those of microfabricated field emitters and have room for improvement for practical applications. In the future, they may be improved by lowering the work function of graphene by chemical/electrostatic doping or employing other low-dimensional materials with lower work functions as the filaments.2/Si wafer substrates with a SiO2 layer thickness of 300 nm using microfabrication technologies. Graphene films are first obtained by mechanical exfoliation of a thin foil of highly oriented pyrolytic graphite and the films with a thickness of one to three layers as determined from their contrast under optical microscope observation are selected for GME fabrications. The selected graphene films are first tailored to ribbons in pre-designed dimensions and configurations by electron beam lithography (EBL) followed by plasma etching, then metal electrodes (70 nm Au/5 nm Cr) are fabricated using standard EBL, metal film deposition and lift-off processes. To make graphene ribbons suspended, the SiO2 layer underneath them is removed by chemical etching. The SiO2/Si wafer is first coated by a layer of polymethyl methacrylate (PMMA) film acting as the mask and small windows in the PMMA mask are opened by EBL to expose the areas for chemical etching, then the wafer is immersed in buffered hydrofluoric acid to remove SiO2 layer. After removing the PMMA mask and drying the wafer in hot isopropanol, graphene emitters suspended between two metal electrodes and over the bottom highly doped Si electrode are obtained. All GMEs in an array are fabricated from the same original graphene films with uniform thickness.GMEs and GME arrays are fabricated on SiO−3 Pa.Electron emission performances of GMEs in direct current mode are measured on a probe station (Lakeshore TTP4) by using a Keithley 4200 semiconductor characterization system. A W probe is used as the top collector electrode. The temporal response of GMEs is studied inside a FEI Quanta 600 F SEM by using a Kleindiek MM3A probing system to achieve electrical connections. Square wave signal is input by a waveform generator (Agilent 33220A) and the output signal of ETD is detected and recorded by an oscilloscope (Aglient DSO7054A). When measuring the temporal response of GMEs, the electron beam of SEM is blanked, and the grid voltage and scintilator voltage of ETD are set to be 210 and 10,000 V, respectively. All measurements are performed at room temperature and a vacuum level of ∼10How to cite this article: Wu, G. et al. Tunable graphene micro-emitters with fast temporal response and controllable electron emission. Nat. Commun. 7:11513 doi: 10.1038/ncomms11513 (2016).Supplementary Figures 1-11, Supplementary Discussion and Supplementary References


Background. Recent reports indicate that matrix metalloproteinase-7 (MMP-7) and CC-chemokine ligand 18 (CCL18) are potential disease markers of idiopathic pulmonary fibrosis (IPF). The objective of this study was to perform direct comparisons of these two biomarkers with three well-investigated serum markers of IPF, Krebs von den Lungen-6 (KL-6), surfactant protein-A (SP-A), and SP-D. Methods. The serum levels of MMP-7, CCL18, KL-6, SP-A, and SP-D were evaluated in 65 patients with IPF, 31 patients with bacterial pneumonia, and 101 healthy controls. The prognostic performance of these five biomarkers was evaluated in patients with IPF. Results. The serum levels of MMP-7, KL-6, and SP-D in patients with IPF were significantly elevated compared to those in patients with bacterial pneumonia and in the healthy controls. Multivariate survival analysis showed that serum MMP-7 and KL-6 levels were independent predictors in IPF patients. Moreover, elevated levels of both KL-6 and MMP-7 were associated with poorer survival rates in IPF patients, and the combination of both markers provided the best risk discrimination using the C statistic. Conclusions. The present results indicated that MMP-7 and KL-6 were promising prognostic markers of IPF, and the combination of the two markers might improve survival prediction in patients with IPF. Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease of unknown etiology with a median survival of 2-3 years from the time of diagnosis. At present, high-resolution computed tomography (HRCT) is an essential component of the diagnosis of IPF . SurgicaKrebs von den Lungen-6 (KL-6), surfactant protein-A (SP-A), and SP-D are type II pneumocyte-derived molecules which have been investigated by our group and other investigators for their usefulness as serum biomarkers of IPF –6. TheseIn addition to the abovementioned type II pneumocyte-derived biomarkers, recent reports indicate that matrix metalloproteinase-7 (MMP-7) and CC-chemokine ligand 18 (CCL18) are potential diagnostic and prognostic markers of IPF. MMP-7 has been shown to be upregulated in the lungs in IPF, particularly in alveolar macrophages and hyperplastic epithelial cells . BronchoThe aim of this study was to perform direct comparisons of the abovementioned five serum biomarkers as disease markers for IPF. We evaluated the serum levels of the five biomarkers in patients with IPF and control subjects, which consisted of patients with bacterial pneumonia (BP) and healthy controls (HC), and determined the relative values of these biomarkers in discriminating IPF patients from control subjects. Moreover, we examined independent predictive values of serum markers for survival of patients with IPF and tested their additive predictive ability compared with clinical information using the C statistic.Sixty-five patients with IPF, 31 patients with BP, and 101 HC were included in the present study. IPF was diagnosed by clinical features, laboratory findings, chest HRCT, and/or surgical lung biopsy, according to the ATS/ERS/JRS/ALAT statement [1), were performed according to the ATS/ERS recommendation [LCO) was measured by the single-breath method in IPF patients, and lung function measurements were performed at diagnosis in these patients. Reference values were obtained from Japanese reference values for spirometry and DLCO, and the percentages of predicted normal values were calculated.Spirometric measurements, including vital capacity (VC) and forced expiratory volume in 1 second  kits . Serum KL-6 levels were measured by sandwich-type electrochemiluminescence immunoassay (ECLIA) using a Picolumi 8220 Analyzer , as previously described .The results were expressed as the mean ± SD. Demographic characteristics and the levels of serum biomarkers were compared between the subject groups using Bonferroni's test. The levels of serum biomarkers were further analyzed by receiver operating characteristic (ROC) curves to determine the cut-off levels that resulted in the optimal diagnostic accuracy for each marker between the 65 patients with IPF and the 132 control subjects, including BP and HC. The use of these cut-off levels allowed the calculation of sensitivity, specificity, diagnostic accuracy, and likelihood ratio of the five biomarkers for separating the IPF patients from the control subjects. A likelihood ratio above 10 indicates strong diagnostic evidence .p value was < 0.05.In the survival analysis of the IPF patients, another ROC curve analysis was conducted to find an optimal cut-off level for the prediction of 5-year survival. The 5-year mortality between two groups was compared using the Kaplan-Meier method and the log rank test. Univariate and multivariate Cox proportional hazards model was used to identify predictors of 5-year survival in IPF patients. Martingale residuals plots were employed to check for assumptions of the proportional hazards and the linearity of each biomarker. The plots were visually evaluated with the help of locally weighted regression scatterplot smoothing . SubsequThe mean age of the IPF patients was 69.3 years, and the patients with IPF were significantly older than the HC. The mean pack-years of smoking were 37.6 in IPF patients, which was significantly higher than those in the HC. There was no significant difference in age and smoking pack-years between the patients with IPF and those with BP. In the lung function analysis, the mean % VC in patients with IPF was significantly lower than that in the HC .Baseline serum levels of the five biomarkers in patients with IPF were significantly higher than those in the HC. Moreover, serum levels of MMP-7, KL-6, and SP-D in patients with IPF were significantly elevated compared with those in patients with BP. However, there was no significant difference in the serum levels of CCL18 and SP-A between patients with IPF and patients with BP. Moreover, serum levels of MMP-7, CCL18, SP-A, and SP-D were significantly elevated in patients with BP compared with the HC .http://dx.doi.org/10.1155/2016/4759040). Subsequently, we evaluated the discriminative power of each biomarker to differentiate between IPF and BP and between IPF and HC, separately. Only KL-6 showed a high discriminatory ability whereas SP-A and CCL18 were poor indicators for discriminating IPF from BP (Table S1(a) and Figure S2(a)). On the other hand, all five biomarkers were useful to distinguish patients with IPF from the HC (Table S1(b) and Figure S2(b)).ROC curve analysis was used to evaluate the discriminating capability of the five serum biomarkers to differentiate IPF patients from control subjects . Cut-offThe median followup period in IPF was 31.0  months. To find an optimal cut-off level that could discriminate survivors from nonsurvivors, another ROC curve was drawn (figure not shown). Survival in IPF patients using biomarker levels above or below the cut-off level was estimated using the Kaplan-Meier method. Survival was significantly different between higher and lower levels of MMP-7, CCL18, and KL-6 .p = 0.0336) and KL-6  were shown to be independent predictors for 5-year mortality , 1.074; ortality . The fitAs shown in In the present study, we directly compared the diagnostic and prognostic value of five serum biomarkers—MMP-7, CCL18, KL-6, SP-A, and SP-D—in patients with IPF and control subjects. Multivariate Cox analysis showed that serum levels of MMP-7 and KL-6 were independent predictors of prognosis in IPF patients. In addition, IPF patients with elevated levels of both KL-6 and MMP-7 had worse survival rates, and the combination of the two markers with the baseline covariates provided the highest C index. These findings indicated that both MMP-7 and KL-6 were promising prognostic markers of IPF, and a combination of the two markers might improve the survival prediction in patients with IPF. Additionally, we showed that MMP-7 and KL-6 could clearly differentiate IPF patients from patients with bacterial pneumonia and healthy controls, suggesting their potential as diagnostic biomarkers.In this study, MMP-7 and KL-6 were independent predictors of prognosis in patients with IPF, which was consistent with the results of previous reports , 21. MorThe present results indicate that MMP-7 could be a promising diagnostic marker of IPF. The ROC curve analysis showed the excellent discriminative capability of MMP-7, as indicated by the high area under the curve (AUC) value >0.90) and likelihood ratio >10). It should be noted that the ability of a biomarker to discriminate IPF from BP does not necessarily indicate that the biomarker is sufficient, by itself, for diagnosing IPF, although low false positive rates in BP would be an important feature for diagnostic markers for ILDs as shown in our previous study [0 and lik. It shouOur study also demonstrated that serum levels of CCL18 had moderate discriminatory ability for differentiating IPF patients from the HC; however, CCL18 was found to be a poor indicator for distinguishing IPF from BP. An in vitro study reported that CCL18 had a chemotactic effect on lung fibroblasts and stimulated collagen production . HoweverThe present study had several limitations. First, this study is a retrospective review of patients with IPF prospectively recruited from one tertiary hospital, and only Japanese participants who agreed to join this study were included. Therefore, our results may not be generalized to all patients with IPF. Second, there are distinctions in age and smoking histories between patients with IPF and HC. Third, control groups in this study consisted of only patients with BP, an acute lung disease, and healthy subjects; patients with chronic lung diseases, especially other ILDs, were not included in the control group. Therefore, we did not fully evaluate the diagnostic utility of the serum biomarkers. It should be noted that no clinically useful biomarker for distinguishing IPF from other ILDs has been found. We have previously reported that serum levels of KL-6 were also elevated in patients with hypersensitivity pneumonia (HP) . Ishii eOur results showed that both MMP-7 and KL-6 might be a useful prognostic marker of IPF, and a combination of the two markers may improve survival prediction in patients with IPF. Additionally, we showed that MMP-7 and KL-6 could differentiate IPF patients from patients with bacterial pneumonia and healthy controls. These results indicate that the measurement of serum levels of KL-6 and/or MMP-7 could potentially support the diagnosis of IPF and would be useful for identifying vulnerable patients especially when the two markers are used in combination. Further large-scale investigation would be warranted to confirm this finding and to find the best method to use this combination of biomarkers of IPF.Supplementary table 1 and supplementary figure 2 shows the diagnostic value and cut-off levels of the five biomarkers to discriminate patients with IPF from those with bacterial pneumonia and healthy subjects, separately.Supplementary figure 1 shows the ROC curve analysis in serum levels of KL-6, MMP-7 and the combination of serum levels of KL-6 and MMP-7 to distinguish IPF patients from patients with bacterial pneumonia and healthy controls. Supplementary figure 3 shows the Kaplan-Meier analysis of the IPF patients using biomarker levels.Supplementary figure 4 shows the Martingale residuals plots employed to check for assumptions of the Cox proportional hazards model.


Most human microbiota studies focus on bacteria inhabiting body surfaces, but these surfaces also are home to large populations of viruses. Many are bacteriophages, and their role in driving bacterial diversity is difficult to decipher without the use of in vitro ecosystems that can reproduce human microbial communities.We used chemostat culture systems known to harbor diverse fecal bacteria to decipher whether these cultures also are home to phage communities. We found that there are vast viral communities inhabiting these ecosystems, with estimated concentrations similar to those found in human feces. The viral communities are composed entirely of bacteriophages and likely contain both temperate and lytic phages based on their similarities to other known phages. We examined the cultured phage communities at five separate time points over 24 days and found that they were highly individual-specific, suggesting that much of the subject-specificity found in human viromes also is captured by this culture-based system. A high proportion of the community membership is conserved over time, but the cultured communities maintain more similarity with other intra-subject cultures than they do to human feces. In four of the five subjects, estimated viral diversity between fecal and cultured communities was highly similar.Because the diversity of phages in these cultured fecal communities have similarities to those found in humans, we believe these communities can serve as valuable ecosystems to help uncover the role of phages in human microbial communities.The online version of this article (doi:10.1186/s40168-015-0124-3) contains supplementary material, which is available to authorized users. Human body surfaces are inhabited by diverse viral communities, and the majority of those identifiable viruses are bacteriophages , 2. The Bacteriophages possess the capacity to alter microbial communities by either lysing their hosts or providing phenotypic advantages to recipient bacteria –15. PhagStudies of microbiota can be limited due to restrictions imposed by working with human subjects . For exaModel microbial systems must be reproducible, highly stable, and must retain similar levels of diversity to the inocula from which they were derived , 28. Int9 FSPs in feces and 1.4 × 109 FSPs in the chemostat cultures for all subjects studied. The presence of such high densities of FSPs in both sample types strongly suggested the presence of substantial viral communities.We recruited five human subjects through the University of Guelph and sampled their feces. Donors #1, #2, and #10 were a co-habiting family unit of father, mother, and child, respectively, while donors #8 and #9 were unrelated. Each fecal sample was homogenized and processed immediately into a chemostat vessel, and the remainder of the feces was stored until processing of the viromes could take place. Chemostat vessels were operated under conditions designed to mimic the human distal colon . Culture−5) and found that all were free of 16S rRNA gene homologues. We also used BLASTN to search the viromes for homologues against a human reference genome, and some similar sequences  were identified and removed prior to further analysis. These data suggest that these chemostat and fecal viromes were relatively free of contaminating cellular nucleic acids.We isolated and processed viruses from both feces and chemostat cultures utilizing sequential filtration followed by cesium chloride density gradient centrifugation according to our previously described protocols . We sequp = 0.01)   to determine the relative proportion of our viromes that had significant sequence similarities. We utilized the numbers of reads assigned to each contig to identify the proportions of reads belonging to contigs with significant sequence similarities in the NR database. We found that in all subjects combined, 94.9 ± 1.7 % of the reads belonged to contigs that had similar sequences in the NR database  from donor #1 Fig.  and 1981We also analyzed the cultured viral communities using the MG-RAST Server  and cateWe identified significant sequence similarities to each assembled viral contig by BLASTX analysis against the NCBI non-redundant database to decipher which viral genes had similarities in the chemostat cultures. The vast majority of the contigs had similarities to hypothetical phage proteins, proteins involved in replication/integration, restriction/modification enzymes, or tail fibers . Similar results were found for fecal communities, where 25 % of the viruses sampled across subjects had significant sequence similarities compared with only 13 % when comparing between cultured communities and fecal communities (p = 0.141).We previously demonstrated that phages in the mouth are highly persistent members of human oral microbial communities . We utilp ≤ 0.05) trend observed, where the viruses in each of the cultured communities were significantly individual-specific.We created assemblies from all contigs in each donor to determine the relative proportions of viral contigs that were conserved in each donor over time. In donor #1, 53.3 ± 4.0 % of the contigs were conserved amongst the chemostat cultures over time compared to 25.9 ± 3.0 % conserved between the chemostat cultures and the feces  based on the beta diversity between the fecal and cultured viromes to determine if there were patterns of variation specific to each subject and sample type. Each of the cultured viromes reflected the subject from which they were derived Fig. . While tWe developed tools for characterizing viral communities to discern whether richness and diversity were conserved between fecal and cultured communities. The technique, termed the Homologous Virus Diversity Index (HVDI), is primarily based upon the Shannon Index  and is uWe next used the HVDI to perform rarefaction analysis to determine whether the viruses in the viromes had been adequately sampled and as a measure of whether the richness of viruses differed substantially between fecal and cultured communities. In this case, we calculated the HVDI based on the Chao1 index  because We next compared the results of the HVDI using the Shannon Index to investigate whether the diversity of the viral component of cultured communities was similar to that from the feces in each subject. We found that for all subjects, viral diversity in the cultured communities changed as a function of time. For example, in donor #2, viral diversity generally increased from day 4 to day 24, while viral community diversity generally decreased in donor #9. For donors #1, #2, and #10, the diversity present in cultured communities on day 24 was highly similar to that in the feces of each subject Fig. . For donHuman body surfaces are home to large populations of viruses, whose role as members of human microbial communities is not well understood. In some environments, viral communities have been shown to be involved in driving bacterial diversity, yet no such data exists for human viral communities. We know that many of the phages that populate these communities are highly persistent and carry numerous pathogenic gene functions such as antibiotic resistance, which could help to shape bacterial community membership and its response to certain perturbations. Measuring their effects in humans is not without its challenges, which include the need to use drugs such as antibiotics, the need to reduce confounders such as diet variability, and the need to improve compliance with study protocols. The development of ecosystems that can approximate the dynamic interactions between phage communities and their cellular hosts can greatly reduce the reliance on human subjects in the characterization of human microbial communities.Each virome in this study was subjected to MDA amplification due to the small amounts of viral DNA recovered from each donor. MDA amplification is known to introduce biases into sequence data , and it Different bacteriophage families often have different lifestyles. Because phages can be significant drivers of diversity in different ecosystems , 39, 50,Because of the high diversity in human phage communities and the varying relationships between host and phages, the relative abundances of phages do not necessarily reflect those of their host bacteria. This phenomenon was demonstrated in a prior study of human oral viral communities  and in aOur prior studies of human viral communities have demonstrated that diversity in these communities is generally overestimated . In thatOne of the major goals of this work was to examine how closely cultured communities might approximate human indigenous phage communities. Identifying numerous FSPs and many reconstructed phages in the cultured communities Figs.  suggest By establishing phage communities that have some similarities to those found on human body surfaces, several important questions can be addressed. These questions include the following: (1) what is the role of phages in driving the diversity of the bacteria in human microbial communities?, (2) how do perturbations such as antibiotics impact human phage communities?, (3) how does the sharing of phage communities between individuals impact microbial community membership?, and (4) what are the dynamics of most phages as members of human microbial communities? While there are quantifiable differences between the phage communities present in feces and cultured communities, there also are many similarities. The relative number of FSPs in both sample types are similar, the profiles of beta diversity strongly suggest a conservation of some phage community members across fecal and cultured communities, and the relative levels of phage diversity between communities in some subjects were highly similar. By establishing cultured phage communities, we can begin to understand the role and contributions of phages to human microbial communities.Human subject recruitment and enrollment in this study was approved by The Research Ethics Board of the University of Guelph REB#13AP008 and 10JL002. Each subject signed a written informed consent confirming their willingness to participate in this study.Five healthy donors provided fecal samples: donor #1 , donor #2 , donor #8 , donor #9 , and donor #10 . Donors #1, #2, and #10 were a co-habiting family unit of father, mother, and child, respectively. Donors #8 and #9 were unrelated. None of the donors had a recent history of antibiotic treatment for 9 months prior to sampling. Each donor provided at least 5 g of fresh fecal samples for the chemostat cultures. Donor #10 provided a sample in the home environment which was immediately wrapped in plastic cling wrap to exclude air and then frozen at −20 °C for overnight transportation to the lab. The remaining donors provided fresh samples.2, 5 % CO2, 5 % H2) upon receipt at the lab; for the fresh samples, this was within 30 min of collection. The sample from donor #10 was allowed to thaw in the chamber. A modified Infors Multifors system was used to run chemostat cultures modeling the human distal colon environment, as described by McDonald et al. [w/v) slurry was prepared from each donor by homogenizing feces in pre-reduced growth medium using a stomacher. For every 1 L of medium, the following components were included in the growth medium: peptone water, 2 g; yeast extract, 2 g, NaHCO3, 2 g; CaCl2, 0.01 g; pectin (from citrus), 2 g; xylan (from beechwood), 2 g; arabinogalactan, 2 g; starch , 5 g; casein, 3 g; inulin (from Dahlia tubers), 1 g; bile salts, 0.5 g NaCl, 0.1 g; L-cysteine HCl, 0.5 g; K2HPO4, 0.04 g; KH2PO4, 0.04 g; MgSO4, 0.01 g; hemin, 0.005 g; and menadione, 0.001 g . Growth media was stored at 4 °C until use for a maximum of 2 weeks. The fecal slurry in growth medium was then centrifuged to remove large particles [−1 with constant sparging of O2 free N2 gas to maintain positive pressure and anaerobiosis. Each chemostat vessel was sampled daily by aseptically removing 4 mL of culture directly from the vessel contents, and all samples were archived at −80 °C. Aliquots of the original fecal samples also were archived at −80 °C for virome processing.All samples were placed immediately into an anaerobic chamber  of the supernatant from each donor was resuspended in 190 μL of 0.02-μm filtered PBS and their counts per milliliter determined by epifluorescence microscopy [Fecal viromes were prepared by diluting 0.4 g of feces in 4 mL of SM buffer. The fecal samples were vortexed vigorously for 40 min to separate viral particles, spun at 4000×croscopy . Chemostcroscopy . Only thcroscopy  was reta−5) against the Ribosomal Database Project 16S rRNA genes database [ftp://ftp.ncbi.nlm.nih.gov/genomes/H_sapiens/. Any reads with significant sequence similarities to human sequences were removed prior to further analysis. Length and GC content variation amongst contigs was assessed using Box and Whiskers plots created using Microsoft Excel 2007 . All reads were assembled using CLC Genomics Workbench 8.01 based on 98 % identity with a minimum of 50 % read overlap, which were more stringent than criteria developed to discriminate between highly related viruses [www.phantome.org; ftp://ftp.ncbi.nih.gov/genomes/Viruses/) was performed using CLC Genomics Workbench 8.01  and were mapped using 98 % identity over a minimum of 50 % of the read length. For each donor, we also utilized a separate technique for assembly by constructing global assemblies from all contigs from all time points using 98 % identity over a minimum of 50 % overlap. Viral contigs were analyzed using FGenesV  for ORF prediction and individual ORFs analyzed using BLASTP analysis against the NCBI non-redundant database . If the best hit was to a gene with no known function, lower level hits were used for the annotation as long as they had known putative function and still met the E-value cutoff (10−5).Due to the error rate of semiconductor sequencing , we trimdatabase  and the  viruses . The ass viruses . We also viruses  and IDBA viruses  in the c−5. Specific viral sequences were identified by parsing BLASTX results for known viral genes including replication, structural, transposition, restriction/modification, hypothetical, and other genes previously found in viruses for which the E-value was at least 10−5. Each individual virome contig was annotated using this technique; however, if the best hit for any portion of the contig was to a gene with no known function, lower level hits were used as long as they had known function and still met the E-value cutoff. The annotation data were compiled by the number of reads used to assemble each contig for each subject and used to determine the relative proportions of contigs that contained viral sequences. The phyla of the BLASTX best hits for each annotated contig were used to create profiles in each donor and sample type. We utilized the average coverage in the assemblies of each contig to determine the relative abundance profiles of different phyla to compensate for viruses that may be more abundant than others. This technique prevented reads involved in the assembly of the same virus contigs from being assigned to different putative host phyla based on different BLASTX similarities. Determination of the relative abundances of virus families was determined by BLASTX analysis of the SEED database using MG-RAST [Contigs were annotated using BLASTX against the NCBI NR database with an E-value cutoff value of 10 MG-RAST .−10). Principal coordinates analysis (PCOA) was performed on virome contigs with Bray Curtis distances using Qiime [Analysis of shared sequence similarities present in each virome was performed by creating custom BLAST databases for each virome, comparing each database with all other viromes using BLASTN analysis . We amplified the bacterial 16S rRNA gene V1-V2 hypervariable region using the forward primer 8F fused with the Ion Torrent Adaptor A sequence and one of 23 unique 10 base pair barcodes and reverse primer 357R fused with the Ion Torrent Adaptor P1 from the each donor and sample type . Resultip value was computed as the fraction of times the simulated statistic for each exceeded the observed statistic. For analysis of fecal versus cultured viromes, a randomly chosen donor from the cultured virome group was compared with a randomly selected donor from the fecal group to determine the null distribution of the fraction of shared contigs. We then estimated the fraction of shared sequences with significant similarities from randomly chosen donors within the cultured virome group and compared with the empirical null distribution from simulated inter-group values. Intra-subject comparisons were excluded from this analysis. We estimated the p value based on the fraction of times the intra-group statistic exceeded that for the null statistic.To assess whether virome contigs had significant overlap within or between donors and sample types, we performed a permutation test based on resampling . We simulated the distribution of the fraction of virome sequences with significant similarities from two different sample types within a donor. For each set, we computed the summed fraction of sequences with significant similarities using 1000 random contigs between different donors, and from these computed an empirical null distribution of our statistic of interest (the fraction of shared similar sequences). The simulated statistics within each donor were referred to the null distribution of inter-donor comparisons, and the ftp://ftp.ncbi.nlm.nih.gov/genomes/Viruses/) and Phantome viral databases (www.phantome.org). First, a random set of viruses was chosen from amongst the databases for each virome, and viromes were constructed that contained 10 viruses, 50 viruses, 100 viruses, 500 viruses, and 1000 viruses. We constructed 10 different viromes at each level of different viral genotypes. The reads in each virome were of mean length 200 bp and were chosen randomly across the genomes of each virus. The shortest reads were 150 bp, and the longest reads were 250 bp in each constructed virome. Each virome also was constructed to meet specific evenness requirements, where an evenness of close to 1 would indicate a population consisting of different viruses all of similar relative abundances, and an evenness close to 0 would indicate a population where the relative abundance of a few viruses are much greater than others. We utilized evenness as criteria to construct the viromes because higher evenness values would prove more difficult for the assembly process. For each constructed virome, we created contig spectra by counting the number of reads assigned to each different virus in the virome. The contig spectra then were used as surrogates for population structures in determination of the Shannon Index [−20) over 50 % of the length of the shorter contig were assigned to the same viral genotype. The spectra from each individual contig that were assigned to the same genotype were added to a corrected contig spectrum for each subject and those spectra used as inputs for the Shannon Index [To measure alpha diversity in the viral communities, we utilized a technique termed the Homologous Virus Diversity Index . The tecon Index . The acton Index  to calcuon Index , which uAll sequences including viromes and 16S rRNA genes are available for download in the MG-RAST database (metagenomics.anl.gov/) under the project “Chemostat,” or project #10563.


Programmed cell death-ligand 1(PD-L1) was expressed in various malignancies, and interaction with its receptor programmed cell death 1 (PD-1) often contributed to immune evasion of tumor cells. In this study, we explored the expression of PD-L1 and its correlation with clinical outcomes in gliomas.Clinicopathological data of 229 patients with gliomas was collected. PD-L1 expression was assessed by tissue-microarray-based immunohistochemistry. Over 5% of tumor cells with cytoplasm or membrane staining was defined as PD-L1 positive expression. The associations of clinicopathological features with overall survival (OS) and disease-free survival (DFS) were analyzed by univariate analysis and multivariate analysis was further performed by Cox regression model.PD-L1 positive expression was observed in 51.1% gliomas patients and no significant association was verified between PD-L1 expression and pathological grade in 229 gliomas patients. However, PD-L1 expression rate was 49.2%, 53.7% and 68.8% for grade II, III and IV in 161 patients with those ≥ 12 months of OS, respectively. Although no significant discrepancies was displayed, there was a certain degree of differences between PD-L1 expression and pathological grade . Univariate analysis showed that PD-L1 expression was significantly associated with poor OS in the patients with long-time survival or follow up (OS ≥ 12 months) (P = 0.018), especially in patients with grade IV (P = 0.019). Multivariate analysis revealed that a strong tendency towards statistical significance was found between PD-L1 expression and poor OS (P = 0.081).In gliomas patients with long-time survival or follow up, PD-L1 positive expression could indicate the poor prognosis and it is possible that immunotherapy targeting PD-L1 pathway needed to be determined in the further study. Gliomas are the most common brain tumor and they were categorized into low-grade gliomas , and high-grade gliomas  according to the following criteria including cell density, cell atypia, mitoses and presence or absence of necrosis . The conIn the past decades, molecular targeted therapies are beneficial to improve the prognosis of gliomas, such as bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF) , nimotuzProgrammed cell death-1 (PD-1)/Program death-ligand 1(PD-L1) pathway is a classic immune checkpoint of promising immunotherapeutic strategies. PD-L1 could help tumor cells immune evasion in combination with immunomodulatory properties . BlockagThe clinicopathological features of 229 patients were demonstrated in Table In the study, the correlation of PD-L1 expression with the clinicopathological features was demonstrated in Table Univariate analysis showed that PD-L1 expression was not significantly correlated with OS  was achieved for the retrospective analysis. Part of the enrolled patients was dead. Therefore, it was not deemed to be necessary by the ethics committee, and all samples were anonymous.In the study, we collected information for clinicopathological features and survival time in patients with gliomas, 229 samples were achieved in Sun Yat-Sen University Cancer Center from January 2000 to August 2008. The screening of specimens was suitable for the following conditions: gliomas was diagnosed by 2008 World Health Organization classification of central nervous system tumors; The complete clinicopathological data included age, gender, tumor site, histological subtype, pathological grade; neither previous malignancies nor an secondary tumor; without neoadjuvant chemotherapy (IC), radiotherapy (RT) and chemoradiotherapy (CRT) before surgical resection, follow-up regularly. The Institute Research Medical Ethics Committee of Sun Yat-Sen University granted approval for this study.Tissue microarrays were constructed in accordance with a previously described method . In brieFormalin-fixed, paraffin-embedded gliomas samples were cut into 4-μm thick sequential sections and processed for IHC according to the previously described protocol . The TMAThe expression status of PD-L1 was evaluated by microscopic observation of stained TMA slides. The staining of cytoplasm or membranes was deemed to be positive for PD-L1 expression in over 5% tumor cells . PD-L1 eStatistical analysis was performed with SPSS software, version 16.0 . The correlation between PD-L1 expression and clinicopathological features was assessed by chi-square test. Kaplan–Meier method was used to evaluate the relationship between survival time and clinicopathological parameters. Survival time included overall survival  and disease-free survival . The Cox proportional hazards regression model was used to define independent prognostic biomarkers that inversely impacted OS and DFS. A two-tailed P-value less than 0.05 was served as statistically significant.