Neuregulin 1 (NRG1) and NMDARs play important roles in various neuronal functions including neural development. NMDARs also promote many cellular events such as proliferation and survival of neuroblasts before synapse formation. Although many recent studies have indicated that NRG1 regulates NMDAR function in cortical neurons, the effect of NRG1 on NMDAR activation before synapse formation is not well studied.A receptor-mediated TrkB activation. Co-immunoprecipitation and proximity ligase assay showed that TrkB interacts with ErbB4 (NRG1 receptor) and the TrkB-ErbB4 interaction was increased following NRG1 treatment. A significant reduction in TrkB-ErbB4 interaction was observed in the prefrontal cortex of schizophrenia subjects. We found significant increase in released BDNF levels following NRG1 treatment, which was inhibited by ErbB4 inhibitor, AG1478. In addition, pretreatment with BDNF neutralizing antibody, but not control IgG abolished NRG1-induced increases in phospho-TrkB and phospho-NR2B levels. Moreover, studies using TrkB mutants showed that intercellular domain of TrkB is necessary for TrkB-ErbB4 interaction and NR2B activation.NRG1 induces activation of NMDAR subunit NR2B, and tropomyosin-related kinase receptor B (TrkB), the receptor for BDNF via activation of phospholipase C-gamma (PLC-γ) in immature primary cortical neurons. Our data using TrkB inhibitor (K252a), TrkB siRNA and TrkB−/− neurons demonstrated that TrkB inhibition suppresses NRG1-induced NR2B activation in neurons. We found that NRG1 stimulation leads to GABABDNF/TrkB signaling plays an important role in the NRG1-stimulated NR2B regulation. These findings could be of relevance to many neurodevelopmental disorders, as NRG1 and BDNF signaling pathways have been implicated in autism and schizophrenia. Activation of NMDA type glutamate receptors (NMDARs) facilitate a number of signaling pathways involved in neuronal development, learning, and memory . The dev2+ signaling is known to mediate NRG1-induced NMDAR regulation in neurons . We. We24]. AR-mediated release of BDNF from neurons, which then act on TrkB-expressing neurons promoting NR2B activation via PLCγ . We found that NRG1 treatment stimulates TrkB phosphorylation in neurons, which was inhibited by a BDNF neutralizing antibody, and BDNF inhibition abolished NRG1-induced increase in TrkB-ErbB4 interaction. NRG1-induced ErbB4 activation is known to increase GABA release . Our finBoth NRG1 and BDNF play important role in neurodevelopment and synaptic plasticity. Since NRG1 and BDNF are also present in non-neuronal cells such as oligodendrocytes and astrocytes, it is important to identify whether the processes similar to the ones found in the current study are functioning in those cell types. Moreover, it will be interesting to examine whether TrkB-ErbB4 interaction plays any role in GABAergic function in interneurons. The signaling mechanism described in this study, in which the regulation of NR2B activation by NRG1 is mediated by BDNF/TrkB signaling, may function in developing neural networks to enable NRG1 to modulate synaptogenesis, and growth of dendrites and axons prior to the formation of functional synapses. It will also be important to determine the extent to which this mechanism persists in the adult nervous system and contributes to the regulation of synaptic plasticity and cognitive function by NRG1 and BDNF. Given the alterations in NRG1 and BDNF signaling pathways result in neuronal dysfunctions as well as the implication of NMDARs in neuroplasticity, our findings on the role of TrkB in NRG1-stimulated NR2B phosphorylation could be of relevance to many neurodevelopmental disorders, as NRG1 and BDNF signaling pathways have been implicated in autism and schizophrenia.−/−) were provided by Dr. Barbara Rohrer, Medical University of South Carolina, Charleston, SC and the colony was maintained in our animal housing facility at the Georgia Regents University. All experiments were done in compliance with Georgia Regents University animal welfare guidelines.Timed pregnant CD-1 mice were purchased from Charles River Laboratories . TrkB knockout  by PCR of tail biopsy DNA . PCR reaction was performed over 35 cycles using GoTaq® Green Master Mix (Promega). Primers utilized were as follows: trkb-n2: 5′-ATGTCGCCCTGGCTGAAGTG; trkbc8: 5′-ACTGACATCCGTAAGCCAGT; pgk3-1: 5′-GGTTCTAAGTACTGTGGTTTCC. Annealing temperature was set at 60°C. The products of the PCR reaction were visualized using agarose gel electrophoresis.TrkBA receptor antagonist, picrotoxin (Tocris) at 100 μM. Inhibitor, agonist or antagonist was added 20 or 30 min prior to NRG1 treatment.Primary cortical neurons were prepared at E16. Neurons were cultured in Neurobasal medium containing, B27 supplement, 10% Fetal Bovine Serum (FBS), penicillin/streptomycin mixture of antibiotics and Glutamax for 4 days. The following pharmacological treatments were used: Neuregulinβ1  at 5 nM ; the Trk4 , anti-pErbB4 , anti-TrkB , anti-pTrkB  , an, an4  and anti-PV staining was performed overnight at 4°C followed by rinsing with PBS and incubation for 1 hr at room temperature with Cy2 and Cy3 conjugated secondary antibodies (Jackson Immunoresearch). Cells were then washed and mounted using ProLong® Gold Antifade Mountant containing DAPI (Molecular Probes). Images were taken using a LCM confocal microscope (Zeiss).In Situ reagents (Sigma). The cortical neurons (~2 × 104 cells/well) were seeded into 24-well plate on poly-D-lysine coated cover slip. After treatment, the cortical neurons were washed twice with ice-cold 1 × PBS and fixed with 4% paraformaldehyde and 120 mM sucrose in PBS at room temperature for 20 min. After permeabilization the cells were incubated in the blocking buffer (provided with the kit) overnight at 37°C in a humidified chamber. The cells were incubated with the primary antibodies, anti-TrkB  and anti-ErbB4  diluted in the antibody diluents for 2 hours at room temperature followed by washing in Buffer A (supplied with the kit) 3 times for 15 minutes and incubation with the PLA probes for one hour at 37°C in a humid chamber. The antibodies were omitted in the PLA control group. The cells were again subjected to a 10 minute wash and a 5 minute wash in Buffer A. The ligation reaction was carried out at 37°C for one hour in a humid chamber followed by a 10 and 5 minute wash in Buffer A. The cells were then incubated with the amplification-polymerase solution for two hours at 37°C in a darkened humidified chamber. After washing with 1x Buffer B (supplied with the kit) for 10 minutes followed by a 1 minute wash with 0.01X buffer B the cells were mounted using the mounting media supplied with the kit. Images were collected using Zen 2012 lite imaging software from several fields of view per experiment. The number of PLA signals per cell (indentified as red spots) was counted from three Z-plane images using ImageJ (NIH).The PLA was performed using Duolink Brain-derived neurotrophic factor was measured with a conventional sandwich ELISA using the BDNF Emax Immuno-Assay System  according to the protocol of the manufacturer.pBiFC-TrkB-FL and pBiFC-TrkB-∆-ICD constructs were kindly provided by Dr Maruyama, Okinawa Institute of Science and Technology, Japan . These cWe used 19 nt siRNA  named Ntrk2 for mouse TrkB and 19 nt siRNA (GCAAGAAGUUCCUCCAGUA) for mouse PLCγ (M-040978-01-0005) from Dharmacon Research Inc. The mouse control siRNA used was 19 nt quadruplex with two 3′ overhanging nucleotides . Transfection of both siRNAs (50 nM) was performed in cultured cortical neurons using Amaxa 4D-Nucleofector Protocol (Lonza) 48 h before the NRG1 treatment.We obtained postmortem prefrontal cortex samples from schizophrenia (N = 15) and control (N = 15) subjects from the Human Brain and Spinal Fluid Resource Center . Description on the demographic details of samples is published elsewhere . The samp) values of less than 5% were considered significant.Quantified data are presented as mean ± SEM and analyzed by GraphPad PRISM. Statistical comparisons between two groups were made using t tests. Comparisons among multiple groups were made using one-way or two-way ANOVA, with Bonferroni’s post hoc analyses to identify significant differences between groups. The probability (The authors declare that they have no competing interests.AP designed the study. CDP performed the experiments and analyzed the data. AP wrote the manuscript. Both authors approved the final version of the manuscript.


The limitations of our cognitive resources necessitate the selection of relevant information from the incoming visual stream. This selection and prioritizing of stimuli allows the organism to adapt to the current conditions. However, the characteristics of this process vary with time and depend on numerous external and internal factors. The present study was aimed at determining how the emotional state affects effective connectivity between visual, attentional and control brain areas during the perception of affective visual stimuli. The Directed Transfer Function was applied on a 32-electrode EEG recording to quantify the direction and intensity of the information flow during two sessions: positive and negative. These data were correlated with a self-report of the emotional state. We demonstrated that the current mood, as measured by self-report, is a factor which affects the patterns of effective cortical connectivity. An increase in prefrontal top-down control over the visual and attentional areas was revealed in a state of tension. It was accompanied by increased outflow within and from the areas recognized as the ventral attentional network. By contrast, a positive emotional state was associated with heightened flow from the parietal to the occipital area. The functional significance of the revealed effects is discussed. The processing of emotional stimuli is a complex and non-linear multi-stage process. On different levels, it is affected by both characteristics of the stimuli itself and numerous cognitive and affective factors such as expectations, cognitive set, previous experience, current mood, or learned strategies of emotional control , focuses on the information that is relevant to the current task or goal-related requirements. It represents top-down control over the perceptual system and is responsible for both spatial and feature-related attention. Anatomically, it is identified as the dorsal fronto-parietal network and is composed of bilateral frontal eye fields (FEF) and intra-parietal sulci with superior parietal lobules  applied on the EEG recording  was expected as a marker of facilitation of bottom-up processes and simultaneous inhibition of top-down attentional control associated with the increased role of stimuli salience. Finally, it was posited that negative stimuli – biologically relevant as they signal potential danger and thus require immediate responses – would reveal stronger effects than those observed for positive ones.Additional computations were performed using prestimulus alpha power over the visual cortex to integrate the connectivity analysis with classic EEG methods. Its level during an idle period, just before the presentation of the stimulus, is thought to reflect the active top-down influence that modulates the sensitivity of the perceptual system  and approved by the Ethical Committee of the Institute of Psychology, Jagiellonian University. Informed consent was obtained from all subjects. Forty-two undergraduate student volunteers , participated in the study after signing a written consent form. All of them were medication-free with no reported history of neurological and psychiatric diseases – or of substance abuse – and had normal or corrected-to-normal vision. During the procedure, the subjects were seated in an air-conditioned soundproof cabin before a 24″ LCD monitor.There were two randomly sequenced sessions, one positive and one negative, which were determined by the valence of the slides displayed. Forty emotional pictures were chosen from the International Affective Picture System (IAPS) database . Based on EEG montage brain atlases , Tension Arousal  and Hedonic Tone (HT: a pleasant and enjoyable state).Next – after ensuring normal distributions of data by means of a series of Kolmogorov-Smirnov tests – the relationships between the subjective scales and DTF values were estimated separately in positive and negative sessions using Pearson’s r correlations. Hence, flow increases or decreases refer to positive or negative correlations respectively, with the subjective scales given observed for the extracted epochs. Finally, because there were multiple comparisons involved, the False Discovery Rate (FDR) procedure  and lower energetic arousal (EA), as well as with more negative valence  were seen as increased flow from the CP6 to CP2 electrode for both sessions, and from the CP2 to the O1 electrode only in the negative session. Detailed correlation coefficients with corresponding p-values (both raw and FDR-adjusted) are shown in Tables p = 0.005), while the positive one revealed no significant relationships.Significant negative correlation between the alpha power and average DTF from the left prefrontal cortex to the visual area was observed in the negative session , as a scale strongly linked to negative valence, turned out to be the most influential factor affecting the strength of connectivity. Some results for Hedonic Tone (HT), which, on the other hand, is strongly linked to positive valence, were also observed. In contrast, Energetic Arousal (EA) did not yield any significant results. Since the EA scale is associated mostly with the energetic and not the valence aspect of the emotional state  as well as to the visual cortex (O/PO electrodes). This vast increase in signal propagation can be interpreted as a heightened top-down influence enforced on the attentional and perceptual systems. Such a pattern could possibly underlie mood-related sensitization to potential threat signals, which can be referred to as “hyper-vigilance”. Indeed, the strengthening of top-down connections is suggested as a phenomenon accompanying the prioritized processing of affective content  as a control center. It has been suggested that the left DLPFC plays a dominant role in continuously monitoring the demands of the current situation and imposing automatic control over the attentional systems  to certain other PFC regions. This was also accompanied by strengthening of connectivity with the right TPJ (located under the CP6 electrode). Importantly, these effects were visible for both sessions, which indicates that they may appear prior to full stimuli evaluation as a tonic sensitization to potential changes in the environment. Indeed, the role of the right IFG in modulating different features of visual attention by influencing the occipital and parietal areas has been widely shown  to the CP2 electrode (over the right IPS). Thus the hypothesis that the path of communication between both attentional networks involves the right TPJ as an interface  was found to be associated with a heightened flow from the electrodes over the right IPS to the occipital area. This effect possibly relates to the extension of the spatial scope of attention in a positive mood, which is accompanied by a simultaneous decrease in sensitivity to negative signals. Some data suggest the causal role of the right IPS in regulating this trade-off by strengthening the top-down spatial features of attention . This would seem to be a plausible neural mechanism underlying the sensitization of the stimuli-driven attention. On the other hand, positive mood  was related to increased flow from the right parietal area to the visual cortex, which could underlie changes in attentional scope.Most importantly, we managed to demonstrate that the current mood, as measured by self-report, is a factor that affects the patterns of effective cortical connectivity. A multiple-item checklist tool was chosen for use in the study, which was intended for more implicit mood measurement, where the researcher’s intentions are not easily disclosed. The intermixing of emotional stimuli with adjectival items could be less susceptible to the influence of demand characteristics, since the consideration of possible experimental expectations in subjects is limited when they are focused on a pending procedure. These factors suggest that the accuracy of such quasi-simultaneous measurement is possible and better than a retrospective check (Hurlburt and Heavey Some other limitations and future directions should also be mentioned. Firstly, the limited spatial accuracy of EEG should be borne in mind when interpreting the results. In this way the proposed correspondence between electrodes and cortical areas should be treated as putative but not certain. Secondly, the indirect inference about attentional characteristics was based on existing theoretical models that link the emotional state with changes in perception. Further verification of the results is recommended in future studies, where different task demands and attentional manipulation can be applied.


Melanin-concentrating hormone (MCH) regulates food intake through activation of the receptor, MCHR1. We have identified AMG 076 as an orally bioavailable potent and selective small molecule antagonist of MCHR1. In mouse models of obesity, AMG 076 caused a reduction in body weight gain in wild-type (MCHR1+/+) but not in knockout (MCHR1−/−) mice. The body weight reduction was associated with decreases in food intake and increases in energy expenditure. Importantly, we show that these MCHR1-dependent effects of AMG 076 were also reflected in improved metabolic phenotypes, increased glucose tolerance and insulin sensitivity. Preliminary data on effects of AMG 076 in obese cynomolgus monkeys are also presented. Obesity has become a global health epidemic affecting people of virtually all ages and socioeconomic groups. Obesity contributes to the deaths of ∼300,000 adults in the US each year, making it one of the leading causes of preventable mortality , a neuropeptide highly conserved among vertebrates, plays an important role in the regulation of food intake and energy balance in mammals. MCH is synthesized in the hypothalamus and zona incerta Nahon  of the bOnly one MCH receptor, MCHR1, is expressed in rodents. MCHR1 was identified as a G protein-coupled receptor -11-Methyl-9-trifluoromethyl-1,3,4,4a,5,6,11,11a-octahydro-pyridocarbazol-2-yl)-ethyl]-cyclohexane carboxylic acid was synthesized at Amgen  was purchased from PerkinElmer  and MCH peptide from Phoenix Pharmaceuticals . Inhibition of MCH-induced Ca2+ mobilization was assayed in HEK293 cells stably expressing human MCHR1 (HEK293-MCHR1), using a FLIPR assay kit from Molecular Devices  according to the manufacturer's protocol. IC50 was determined by nonlinear regression curve fitting program (GraphPad Prism). Inhibition of MCH-induced Ca2+ mobilization in multiple species was measured by aequorin assay in CHO cells transiently expressing MCHR1 of different species. Concentrations of MCH used for the FLIPR and aequorin assays were 12 nM and 35 nM, respectively. All assays were repeated at least three times for Ki and IC50 determination and representative experiments are presented.Radioligand membrane binding and aequorin assays were carried out as described previously .The selectivity of AMG 076 was evaluated in radioligand displacement assays against a panel of 64 GPCRs, transporters and ion channels by Eurofins Panlabs . The affinity of AMG 076 for 5HT2C was also evaluated by Amgen in radioligand displacement-binding assay using 5HT2C membrane and specific radioligand -MCH with a Ki of 0.6 ± 0.10 nM  was associated with a significant increase in measured VO2 compared with the MCHR1(+/+) mice that received vehicle. In contrast, no effect of AMG 076 (100 mg kg−1 day−1) treatment was observed in the MCHR1(−/−) mice compared with the MCHR1(−/−) mice that received vehicle. In summary, these data show that the prevention of high-fat diet-induced body weight gain by AMG 076 is MCHR1-dependent and is at least partly mediated through an increase in energy expenditure.AMG 076 was evaluated for its ability to prevent the accumulation of body fat in C57BL/6 mice transferred onto a high-fat diet. To confirm that the effects of AMG 076 were mediated specifically through MCHR1, MCHR1(−/−) mice with the same genetic background  or sibutramine (10 mg kg−1 day−1). The effect of AMG 076 and sibutramine on body weight was assessed over the period from day 0 to 144  reached statistical significance by day 2, 7, and 3, respectively, and remained statistically significant through day 144. Dose-related exposures of AMG 076 in plasma were confirmed as summarized in Table The diet-induced obese (DIO) mouse model is a well-established animal model for testing anti-obesity agents . Sibutramine also reduced food intake (P < 0.05), but to a lesser extent than AMG 076.Food intake measurements were initiated 3 weeks into the study and continued through day 115 of treatments. A dose-related reduction was observed in daily food intake in response to AMG 076 treatment relative to vehicle during the course of treatment .P < 0.05), was reached only by the 1 mg/kg group. Five of the six monkeys given the vehicle gained significant weight, and at least three monkeys in each of the AMG 076 dose groups showed a commensurate decrease in body weight and BMI. Intra-abdominal fat (IAF) content was measured on day 88 of the treatment using abdominal CT scan and food intake was measured daily. Although a downtrend in IAF and food intake similar to that of body weight and BMI was observed with the AMG 076 treatment groups, the changes did not reach statistical significance by any of the treatment groups  mice treated with AMG 076 up to 100 mg kgWe did not observe significant effect of AMG 076 treatment on food intake in lean mice on high-fat diet. We, however, detected a significant reduction in food intake in DIO mice treated with AMG 076. These observations suggest that different metabolic state of the animals may be critical in determining how antagonism of MCHR1 may contribute to body weight loss, through increasing metabolic rate or suppressing appetite, or both.Obesity has been shown to be a risk factor for type 2 diabetes and is known to exacerbate diabetes in humans (Chen et al. Rodents do not express MCHR2 (Tan et al. A number of MCHR1 antagonists have been developed as potential anti-obesity drugs (Borowsky et al.


We propose a quite concrete approach based on the notion of Köthe-Toeplitz duals with respect to the non-Newtonian calculus. Finally, we derive some inclusion relationships between Köthe space and solidness.The important point to note is that the non-Newtonian calculus is a self-contained system independent of any other system of calculus. Therefore the reader may be surprised to learn that there is a uniform relationship between the corresponding operators of this calculus and the classical calculus. Several basic concepts based on non-Newtonian calculus are presented by Grossman (1983), Grossman and Katz (1978), and Grossman (1979). Following Grossman and Katz, in the present paper, we introduce the sets of bounded, convergent, null series and  It is certainly not unusual to measure deviations by ratios rather than differences. For instance, during the Renaissance, many scholars, including Galileo, discussed the following problem. Two estimates, 10 and 1000, are proposed as the value of a horse, which estimates, if any, deviates more from the true value of 100? The scholars who maintained that deviations should be measured by differences concluded that the estimate of 10 was closer to the true value. However, Galileo eventually maintained that the deviations should be measured by ratios, and he concluded that two estimates deviated equally from the true value. From the story, the question comes out this way, what if we measure by ratios? The answer is the main idea of non-Newtonian calculus which consists of many calculuses such as the classical, geometric, anageometric, and bigeometric calculus.Bashirov et al. , 2 have bs*, cs*, and cs0* consisting of the sets of all bounded, convergent, null series based on the non-Newtonian calculus, as follows:ω* = {x = (xk) : xk ∈ C*for all  k ∈ N}. One can conclude that the sets bs*, cs*, and cs0* are complete non-Newtonian metric spaces with the metric d∞N defined byFollowing Tekin and Başar  we can cbv*, bvp*, and bv∞* of bounded variation sequences in the sense of non-Newtonian calculus, as follows:bv*, bvp*, and bv∞* are complete with corresponding metrics on the right-hand side with (Δx)k = xk ⊖ xk−1, x)k′ = xk ⊖ xk+1 for all k ∈ N.Secondly, we introduce several sets  generator is a one-to-one function whose domain is R and whose range is a subset of B⊆R, the set of real numbers. Each generator generates exactly one arithmetic, and conversely each arithmetic is generated by exactly one generator. As a generator, we choose the function β such that its basic algebraic operations are defined as follows:x, y ∈ R(N), where the non-Newtonian real field R(N)≔{β{x} : x ∈ R} as in [AR} as in .β-positive real numbers, denoted by R+(N), are the numbers x in R such that β-negative real numbers, denoted by R−(N), are those for which beta-zero, beta-one, β(0) and β(1). Further, β-integers turns out to be the following:The X be a nonempty set and let d* : X × X → R(N) be a function such that, for all x, y, z ∈ X, the following axioms hold:  x = y,(NM1)   d* = d*,(NM2)   (NM3)   Then, the pair  and d* are called a non-Newtonian metric space and a non-Newtonian metric on X, respectively.Let X =  be a non-Newtonian metric space. Then the basic notions can be defined as follows. x = (xk) is a function from the set N into the set R(N). The β-real number xk denotes the value of the function at k ∈ N and is called the kth term of the sequence.A sequence xn) in a metric space X =  is said to be ∗-convergent if for every given n0 = n0(ε) ∈ N and x ∈ X such that n > n0 and is denoted by *limn→∞xn = x or n → ∞.A sequence (xn) in X =  is said to be non-Newtonian Cauchy (∗-Cauchy) if for every n0 = n0(ε) ∈ N such that m, n > n0.A sequence  is defined as β(|β−1(x)|) and is denoted by |x|β. For each number x in B ⊂ R(N), x1 and x2 is defined by alpha-generator in  also are the ordered pair of arithmetics (β-arithmetic and α-arithmetic). The sets beta-generator generates beta-arithmetics, respectively. Definitions given for β-arithmetic are also valid for α-arithmetic.Suppose that α-arithmetic is used for arguments and β-arithmetic is used for values; in particular, changes in arguments and values are measured by α-differences and β-differences, respectively. The operators of this calculus type are applied only to functions with arguments in A and values in B. The ∗-limit of a function with two generators α and β is defined byf is ∗-continuous at a point a in A if and only if a is an argument of f and *limx→af(x) = f(a). When α and β are the identity function I, the concepts of ∗-limit and ∗-continuity are identical with those of classical limit and classical continuity.The important point to note here is that α-arithmetic to β-arithmetic is the unique function ι (iota) that possesses the following three properties:(i)ι is one to one.(ii)ι is from A onto B.(iii)u and v in A,For any numbers,  It turns out that ι(x) = β{α−1(x)} for every x in A and that n. Since, for example, α-arithmetic can readily be transformed into a statement in β-arithmetic.The isomorphism from C*; that is,Let C*, ⊕, ⊙) is a field. of ∗-complex numbers, the formal notation,N-series. Also, for integers, n ∈ N, the finite ∗-sums sn* = ∗∑∑k=0nzk* are called the partial sums of complex N-series. If the sequence ∗-converges to a complex number s*, then we say that the series ∗-converges and write s* = ∗∑∑k=0nzn*. The number s* is then called the ∗-sum of this series. If (sn)  ∗-diverges, we say that the series ∗-diverges or it is ∗-divergent.Given a sequence -triangle inequality).(ii)(iii)Let  and zk*, tk* ∈ C* for k ∈ {0,1, 2,3,…, n}. Then, For any Following Tekin and Başar , we can z* ∈ C* be an arbitrary element. The distance function d* is called ∗-norm of z* and is denoted by z1*, z2* ∈ C* we have d* is the induced metric from Let z* by z* but leaves the real part the same. ThusLet (i) Let α and β be the same generator functions and z* ∈ C*. Then the following condition holds(ii) Let C*, d*) is a complete metric space, where d* is defined by l∞*, c*, c0*, and lp*, p ≥ 1, are sequence spaces.The sets (b)λ* denote any of the spaces l∞*, c*, and c0* and z = (zk*), t = (tk*) ∈ λ*. Define d∞* on the space λ* by λ*, d∞*) is a complete metric space.Let (c)l∞*, c*, and c0* are Banach spaces with the norm ||z||∞* defined byThe spaces (d)lp* is Banach spaces with the norm ||z||p* defined byThe space The following statements hold.bs*, cs*, cs0* and bv*, bvp*, bv∞* consisting of all bounded, convergent, null series and the sets of bounded variation sequences in the sense of non-Newtonian calculus which correspond to the sets bs, cs, cs0 and bv, bvp, bv∞ over the complex field C, respectively.In the present section, we introduce the sets μ* denote any of the spaces bs*, cs*, and cs0* and z = (zk*), t = (tk*) ∈ μ*. Define d∞N on the space μ* byα, β operators and corresponding function ι = βα−1. Then,  is a complete metric space.Let cs* and cs0*, we prove the theorem only for the space bs*. Let the ∗-sums ∗∑∑k=0nzk*, ∗∑∑k=0ntk* ∈ C*, where z = (zk*), t = (tk*) ∈ C*. Then the following metric axioms in  (NM1) From  it can b d∞N = d∞N holds.(NM2) It is trivial that the condition  z = (zk*), t = (tk*), w = (wk*) ∈ C*. In fact by taking into account (NM3) We show that ∗triangle inequality in  Since the axioms (NM1)–(NM3) are satisfied,  is a non-Newtonian metric space. It remains to prove the completeness of the space bs*.Since the proof is similar to the spaces xm) be a ∗-Cauchy sequence in bs*, where xm = {x1m) = {xk(1), xk(2),…, xkm) and show that x ∈ bs*. From (r → ∞ and m > n0 we havexm) ∈ bs*, there exists k ∈ N. Thus, (m > n0k ∈ N whose right-hand side does not involve k. Hence (xk) is a bounded sequence of geometric complex numbers; that is, x = (xk) ∈ bs*. Also from (m > n0m → ∞ for an arbitrary ∗-Cauchy sequence (xm). Hence bs* is complete.Let , y = (yk) ∈ bv*, and (Δx)k′ = xk ⊖ xk+1. Then,  is a complete metric space. Similarly, one can conclude that the other bounded variation sets  and  are also complete.Let bv* is a Banach space with the norm ||x||bv* defined byThe space The idea of dual sequence space which plays an important role in the representation of linear functionals and the characterization of matrix transformations between sequence spaces was introduced by Köthe and Toeplitz , whose mλ*, μ* ∈ C*, the set S, defined byλ* and μ* for all k ∈ N. One can easily observe for a sequence space ν* of ∗-complex numbers that the inclusions S ⊂ S if ν* ⊂ λ* and S ⊂ S if μ* ⊂ ν* hold.In this section, we focus on the alpha-, beta-, and gamma-duals of the classical sequence spaces over non-Newtonian complex field. For λ* ⊂ ω* which are, respectively, denoted by {λ*}α, {λ*}β, and {λ*}γ, as follows:wk⊙zk) is the coordinatewise product of ∗-complex numbers w and z for all k ∈ N. Then {λ*}β is called beta-dual of λ* or the set of all convergence factor sequences of λ* in cs*. Firstly, we give a remark concerning the ∗-convergence factor sequences.Firstly, we define the alpha-, beta-, and gamma-duals of a set Throughout the text, we also use the notation “<” for a non-Newtonian linear subspace which was created in .∅ ≠ λ* ⊂ ω*. Then the following statements are valid. λ*}β is a sequence space if φ* < {λ*}β < ω*, where {λ* ⊂ μ* ⊂ ω*, then {μ*}β < {λ*}β.If λ* ⊂ {λ*}ββ : = ({λ*}β)β.φ*}β = ω* and {ω*}β = φ*.{Let w = (wk), m = (mk), and n = (nk)∈{λ*}β. (a)λ*}β < ω* holds from the hypothesis. We show that m ⊕ n ∈ {λ*}β for m, n ∈ {λ*}β. Suppose that l ∈ λ*. Then (mk⊙lk) ∈ cs* and (nk⊙lk) ∈ cs* for all l ∈ λ*. We can deduce thatIt is trivial that { m ⊕ n ∈ {λ*}β. Now, we show that t⊙w ∈ {λ*}β for any t ∈ C* and w = (wk)∈{λ*}β, since (wk⊙lk) ∈ cs* for all l ∈ λ*. Combining this with ((tk⊙wk)⊙lk) = tk⊙(wk⊙lk) ∈ cs* for all l ∈ λ* we get t⊙w ∈ {λ*}β. Therefore, we have proved that {λ*}β is a subspace of the space ω*.Hence, (d)ω*}β ⊂ φ*. Suppose that w = (wn)∈{ω*}β and z = (zn) are given with ∗-division by zn⊙wn = 1* if wn ≠ 0* and zn : = 1* otherwise. By taking into account the set φ* from inclusion (a), then there exists an integer N ∈ N for all n ≥ N such that Using (a) we need only to show { w⊙z ∈ cs* implies that w ∈ φ*. The rest is an immediate consequence of this part and we omitted the details.Further, Since the proof is trivial for the conditions (b) and (c), we prove only (a) and (d). Let c0*}β = {c*}β = {l∞*}β = l1*.{l1*}β = l∞*.{The following statements hold: l∞*}β ⊂ {c*}β ⊂ {c0*}β by l1* ⊂ {l∞*}β and {c0*}β ⊂ l1*. Now, consider that w = (wk) ∈ l1* and z = (zk) ∈ l∞* are given. By taking into account the cases (c)-(d) of w⊙z ∈ cs*. So the condition l1* ⊂ {l∞*}β holds.(a) Obviously {y = (yk) ∈ ω*∖l1*, we prove the existence of an x ∈ c0* with y⊙x ∉ cs*. According to y ∉ l1* we may confirm an index sequence (np) which is strictly increasing with x = (xk) ∈ c0* by xk : = (sgn*yk⊘p), the non-Newtonian complex signum function is defined byy = (yk) ∈ C*. Finally, by using α = β, we getnp−1 ≤ k < np. Therefore y⊙x ∉ cs* and thus y ∉ {c0*}β. Hence {c0*}β ⊂ l1*.Conversely, for a given l∞* ⊂ ({l∞*}β)β = {l1*}β since {l∞*}β = l1*. Now we assume the existence of a w = (wn)∈{l1*}β∖l∞*. Since w is unbounded there exists a subsequence (wnk) of (wn) and we can find a real number (k + 1)2 such that k ∈ N1. The sequence (xn) is defined by xn : = (sgn*(wnk)⊘(k + 1)2) if n = nk and x ∈ l1*. However,w ∉ {l1*}β, which contradicts our assumption and {l1*}β ⊂ l∞*. This step completes the proof.(b) From the condition (c) of ζ-duals .In addition to the statements in ∅ ≠ λ* ⊂ ω*. Then the following statements are valid:φ* < {λ*}α < {λ*}β < {λ*}γ < ω*; in particular, {λ*}ζ is a sequence space over C*;λ* < μ* < ω*, then {μ*}ζ < {λ*}ζ;if I is an index set, if λi* are sequence spaces, and if λ* : = ⋃i∈Iλi*, then 〈λ*〉ζ = ⋂i∈I{λi*}ζ, where the notation "〈〉" stands for the span of linear subspace over C*;λ* ⊂ {λ*}ζζ : = ({λ*}ζ)ζ.Let l∞* < cs* < bs*. We only show the cases (c) and (d) taking ζ = alpha. The rest of the parts can be obtained in a similar way.(c)λi* ⊂ 〈λ*〉 the following 〈λ*〉α ⊂ {λi*}α and 〈λ*〉α ⊂ ⋂i∈I{λi*}α hold by (b). On the other hand, if y ∈ ⋂i∈I{λi*}α, that is, y ∈ {λi*}α, then x⊙y ∈ l1* for all x ∈ λ* and therefore y ∈ {λ*}α ⊂ 〈λ*〉α.Now, as an immediate consequence (d)λ* ⊂ {λ*}ζζ. Let w ∈ λ*; then w⊙z ∈ l1* for all z ∈ {λ*}α; thus w ∈ {λ*}ζζ and λ* ⊂ {λ*}ζζ by (a).We can deduce The case (b) obviously is true, and (a) follows from λ* ≠ {λ*}ζζ as we get from ζ = β and λ* : = c0*. We have {c0*}ββ = l∞* ≠ c0*. This remark gives rise to the following definition.Here ζ ∈ {α, β, γ} and let λ* be a sequence space over the field C*. λ* is called ζ-space if λ* = {λ*}ζζ. Further, an α-space is also called a Köthe space or perfect sequence space.Let From λ* is a sequence space over the field C* and , then {λ*}ζ is a ζ-space; that is, {λ*}ζ = {λ*}ζζζ.If λ*}α = {λ*}β = {λ*}γ. This gives rise to the notion of solidity.Now we look for sufficient conditions for {X be a sequence space. Then X is called solid ifLet λ* be a sequence space over the field C*. Then λ* is solid if and only ifLet u = (uk) ∈ ω* and x = (xk) ∈ λ* with ξk, ηk ∈ R. Obviously the condition ψk, δk ∈ R. We obtain ψk2 + δk2 ≤ ξk2 + ηk2 since k ∈ N. We may choose a real number γk ∈ R with ψk2 + γk2 = ξk2 + ηk2 and consider w = (wk) defined by w ∈ λ* and ζk ∈ R such that ζk2 + δk2 = ξk2 + ηk2 it follows similarly that Let λ* < ω* is any sequence space over the field C*; then the following statements hold. λ* is a Köthe space, then λ* is solid.If λ* is solid, then {λ*}α = {λ*}β = {λ*}γ.If λ* is a Köthe space, then λ* is a ζ-space.If Consider that λ* < ω* be a sequence space over the field C*.(a)λ* is a Köthe space and u ∈ ω*, then u ∈ {λ*}αα if and only if the condition u⊙z ∈ l1* holds for all z ∈ {λ*}α. Besides this we obtain u = (uk) ∈ λ* and v = (vk) ∈ ω* and the statementIf  z ∈ {λ*}α. Therefore v⊙z ∈ l1*. Hence w ∈ λ* and λ* is solid over the field C*.holds for each (b)λ* is a solid sequence space over C*. To show {λ*}α = {λ*}β = {λ*}γ, it is sufficient condition to verify {λ*}γ < {λ*}α in v = (vk) ∈ {λ*}γ; that is,Consider that  λ*, for z = (zk) ∈ λ*, the condition u = (uk) ∈ λ* for all k ∈ N. Therefore by combining this with the inclusion (By taking into account solidness of nclusion  we deduc u⊙v ∈ l1* < cs*. Hence v ∈ {λ*}α and {λ*}γ < {λ*}α.holds and (c)This is an obvious consequence of Let φ*, ω*, lp*, c0*, and l∞* of sequences are solid.The sets c* and bv* of sequences are not solid; therefore none of them is a Köthe space.The sets ζ ∈ {α, β, γ}, thenFor each (i)l1*}ζ = l∞* and {l∞*}ζ = l1*;{(ii)ω*}ζ = φ* and {φ*}ζ = ω*.{ζ ∈ {α, β, γ} and c0* < μ* < l∞*, then {μ*}ζ = l1* and μ* ⊂ {μ*}ζζ = l∞*. In particular, {c0*}ζ = {c*}ζ = l1*, and each of c*, c0* is not a ζ-space.If The following statements hold. c0* and l∞* are solid, we know that {c0*}ζ = {l∞*}ζ = l1*. So the statements in (d) are obtained from That the specified spaces in cases (a-b) are solid is an immediate consequence of their definition. Additionally, the cases (i-ii) of (c) can be obtained ζ-duals of the spaces cs*, bs*, bv*, and bv0*. We will find that none of these sequence spaces is solid; in particular, none of them is a Köthe space.Next, we determine the cs*}α = {bv*}α = {bv0*}α = l1*; {bv0*} = bv*∩c0*,{cs*}β = bv*, {bv*}β = cs*, {bv0*}β = bs*, {bs*}β = bv0*;{cs*}γ = bv*, {bv*}γ = bs*, {bv0*}γ = bs*, {bs*}γ = bv*.{The following statements hold: cs*, bs*, bv*, and bv0* of sequences are β-spaces (ζ = beta), but they are not Köthe spaces. Moreover, the sets bs* and bv* of sequences are γ-spaces (ζ = gamma), whereas both cs* and bv0* are not γ-spaces. None of the spaces cs*, bs*, bv*, and bv0* is solid.In particular, the sets cs*}ζ, ζ ∈ {α, β, γ}, and the proofs of all other cases are quite similar.We prove the cases for the spaces {x = (xk) ∈ cs* and y = (yk) ∈ l1*. Then,x||cs* is defined by (y ∈ {cs*}α which gives the fact that l1* ⊂ {cs*}α.(a) Let fined by . Therefoy = (yk) ∈ {cs*}α∖l1*. Then to every natural number p we can construct an index sequence (np) with np < np+1 and p ∈ N. Define x = (xk) byβ-division β{(−1)k2p−} for np < k ≤ np+1. Then x = (xk) ∈ cs*. According to the choice of np, the inequalitiesp2p diverges. Thus, x⊙y ∉ l1*, which implies y ∉ {cs*}α. This contradicts the fact that y ∈ {cs*}α. Therefore {cs*}α ⊂ l1*.Conversely, suppose that bv0*}α ⊂ l1* holds as well if we take the sequence (xk) byThe condition {u = (uk) ∈ {cs*}β and w = (wk) ∈ c0*. Define the sequence v = (vk) ∈ cs* by vk = (wk ⊖ wk+1) for all k ∈ N. Therefore, ∗∑∑k⊙  vk  ∗-converges, butl1* ⊂ cs* yields (uk) ∈ {cs*}β ⊂ {l1*}β = l∞*. Then we derive by passing to the ∗-limit in  ∈ {c0*}β = {c0*}α = l1*; that is, u ∈ bv*. Therefore, {cs*}β⊆bv*.(b) Let ges, but∑∗ k=0 n u = (uk) ∈ bv*. Then, (uk ⊖ uk−1) ∈ l1*. Further, if v = (vk) ∈ cs*, the sequence (wn) defined by wn = ∗∑∑k=0nvk for all k ∈ N is an element of the space c*. Since {c*}α = l1*, the N-series ∗∑∑kwk⊙(uk ⊖ uk−1) is ∗-convergent. Also, we havewn) ∈ c* and (uk) ∈ bv* ⊂ c*, the right-hand side of inequality ⊙uk or ∗∑∑k=0∞uk⊙vk  ∗-converges. Hence bv*⊆{cs*}β.Conversely, suppose that  we have∑∗ k=m n ∈ {cs*}γ and v = (vn) ∈ c0*. Then, for the sequence (wn) ∈ cs* defined by wn = (vn ⊖ vn+1) for all n ∈ N, we can find a non-Newtonian real number n ∈ N. Since (vn) ∈ c0* and (un)∈{cs*}γ ⊂ l∞*, there exists a non-Newtonian real number n ∈ N. Therefore,uk ⊖ uk−1) ∈ {c0*}γ = {c0*}α = l1*; that is, (un) ∈ bv*. Therefore, since the inclusion {cs*}γ ⊂ bv* holds, we conclude that {cs*}γ = bv*, as desired.(c) By using (a), it is known that Although all arithmetics are isomorphic, only by distinguishing among them, we do obtain suitable tools for constructing all the non-Newtonian calculi. But the usefulness of arithmetics is not limited to the construction of calculi; we believe there is a more fundamental reason for considering alternative arithmetics; they may also be helpful in developing and understanding new systems of measurement that could yield simpler physical laws. average speed. The definition “distance traveled per unit time” is incomplete because it fails to provide a method of determining the average speed of an accelerated particle. The definition “distance divided by time,” though not incorrect, is a gross oversimplification that fails to reveal the underlying issues. Fortunately there is a completely satisfactory definition, which undoubtedly was known to Galileo. Then we isolate a constant in each given uniform motion by defining speed to be the distance traveled in any unit time-interval. Finally, for a particle that moves nonuniformly in a distance d in time t, we define the average speed to be the speed that a particle in uniform motion must have in order to travel a distance d in time t. In our opinion, neither the simplicity nor the obviousness of the answer, d/t, justifies its use as the definition of average speed .Of course, we can only speculate as to future applications of the non-Newtonian calculi. Perhaps they can be used to define new scientific concepts, to yield new or simpler scientific laws, to solve heretofore unsolved problems, or to formulate and solve new problems. For example, one constructs non-Newtonian calculi of functions of two or more real variables by choosing an arithmetic for each axis. It might even be profitable to seek deeper connections among the corresponding operators of the calculi. Like as, some of them are postponed to our future works.


Brain-Derived Neurotrophic Factor (BDNF) is a neurotrophin that generally promotes neuronal proliferation, survival and plasticity. These effects occur through cellular secretion and subsequent binding of BDNF to its cognate receptor, TrkB, which promotes paracrine and autocrine signaling cascades. A BDNF/TrkB autocrine loop has been implicated in the survival of cells representing several human cancers and is associated with poor prognosis. The HTLV-1 protein, HBZ, is localized to the nucleus where it is capable of deregulating gene expression. In this study, we found that HBZ increases expression of BDNF. There are a variety of transcriptional variants of BDNF, and HBZ increased expression of several of these. Furthermore, multiple transcriptional variants of BDNF were upregulated in HTLV-1-infected cells compared to normal CD4 cells. Expression of the TrkB receptor was also higher in HTLV-1-infected cells than in normal CD4 cells. This trend similarly occurred when comparing ATL patient and healthy donor specimens, with patient cells exhibiting higher levels of BDNF and TrkB expression. TrkB appeared to localize to the cytoplasmic membrane of HTLV-1-infected cells, suggesting a BDNF/TrkB loop contributes to the survival of these cells. This hypothesis was confirmed by chemically blocking the binding of BDNF to TrkB or by inhibiting TrkB signaling, as both treatments led to a significant increase in apoptosis. Together these results suggest that HBZ activates a BDNF/TrkB autocrine loop that enhances survival of HTLV-1-infected cells. This mechanism may ultimately facilitate the survival of ATL cells.


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