Delayed comparison tasks are widely used in the study of working memory and perception in psychology and neuroscience. It has long been known, however, that decisions in these tasks are biased. When the two stimuli in a delayed comparison trial are small in magnitude, subjects tend to report that the first stimulus is larger than the second stimulus. In contrast, subjects tend to report that the second stimulus is larger than the first when the stimuli are relatively large. Here we study the computational principles underlying this bias, also known as the contraction bias. We propose that the contraction bias results from a Bayesian computation in which a noisy representation of a magnitude is combined with a-priori information about the distribution of magnitudes to optimize performance. We test our hypothesis on choice behavior in a visual delayed comparison experiment by studying the effect of (i) changing the prior distribution and (ii) changing the uncertainty in the memorized stimulus. We show that choice behavior in both manipulations is consistent with the performance of an observer who uses a Bayesian inference in order to improve performance. Moreover, our results suggest that the contraction bias arises during memory retrieval/decision making and not during memory encoding. These results support the notion that the contraction bias illusion can be understood as resulting from optimality considerations. Comparing magnitudes of two temporally separated stimuli is one of the fundamental tools of experimental psychology and neuroscience. Interestingly, choice behavior in these experiments reveals a fundamental bias: when the first stimulus is small, subjects tend to overestimate it, whereas when it is large, they tend to underestimate it. The first account of this bias, known as the contraction bias, was published a century ago by Harry Levi Hollingworth who later became one of the pioneers of applied psychology. Hollingsworth presented subjects with square cards of various sizes for a brief period of time and asked them to memorize their sizes perceived magnitude of a stimulus is a weighted combination of its veridical magnitude and a reference magnitude, such as an average of all contextually relevant stimuli, that serves as an anchor The customary explanation for the contraction bias is that the There is a growing body of literature suggesting that the brain utilizes Bayes' rule to optimally combine information from different sources In order to test this hypothesis, we conducted an experiment in which we instructed subjects to memorize the length of a bar presented on a computer screen and then compare this memorized length to the length of a probe bar. We show that contraction bias depends on the prior distribution of bar lengths, and increasing the uncertainty in the memory of bar lengths enhances the contraction bias, both of which are consistent with the Bayesian hypothesis.after memory encoding and measuring the magnitude of the contraction bias we demonstrate that prior information is introduced during memory retrieval/decision making rather than when the first stimulus is encoded in memory.When within a trial does the Bayesian computation take place? Is the encoded memory biased or does the prior information bias the result of the length comparison? By manipulating uncertainty in the memory of bar lengths Some of the findings presented here have appeared previously in abstract form 1L) for 1 sec and were instructed to memorize its length. After a delay of 1 sec, during which screen remained blank, they viewed a probe bar (2L). The probe bar remained visible on the screen until subjects reported which of the two bars was longer by pressing dedicated keys on the keyboard. The first bar, L1, was drawn from a uniform distribution in the logarithmic scale between 150 and 600 pixels. The difference in length between 1L and 2L varied between −30% and +30%. Both bars were presented at random locations on the screen and no feedback was provided to the subjects on performance on individual trials (See L1>L2’ responses as a function of the difference between the memorized and probe stimuli. The average psychometric curve of the subjects (n = 9) is plotted in L1 and L2 increased.In the standard task , subjectials See . We quanpattern of errors made by subjects depends on the magnitude of the memorized stimulus. When the memorized stimulus is small, subjects tend to make more errors in trials in which the probe stimulus is larger than the memorized stimulus, compared to trials in which the probe is smaller than the memorized stimulus. The opposite behavior is observed when the magnitude of the memorized stimulus is large L1 and L2 in our experiments) relative to the width of the psychometric curve. We used a different approach to overcome this limitation: Unbeknownst to the subjects, we included a subset of trials in which the lengths of the two bars were identical. We term these trials “impossible trials” because there is no correct answer to the question “which bar (L1 or L2) was longer”. Impossible trials are well suited for the analysis of the contraction bias because performance on these trials is independent of the proficiency of individual subjects in distinguishing the difference in the length of the two bars.Our purpose is to quantify the contraction bias in these experiments. Previous studies have demonstrated a contraction bias in delayed comparison tasks by showing that the L1>L2’ as a function of the length of L1 (L1 = L2). Note that despite the fact that L1 and L2 were identical on these trials, subjects reported that L1 was longer than L2 on roughly 60% of the shortest trials  the control region knows that the neural representations of L1 and L2 are noisy, e.g. due to noise in the sensory pathway. Moreover the representation of L1 is noisier than that of L2 because L1 has to be stored in memory, a process that may contribute additional noise to the representation of L1; (2) the control region has information about the marginal distribution of bar lengths. This distribution can be approximated based on the history of the experiment; (3) the control region utilizes Bayes' rule and combines the noisy representations of L1 and L2 with knowledge about the prior distribution in order to construct the posterior distributions for the two bar lengths. These posteriors are then used to minimize error in judgment. A formal description of this process appears in the Our aim is to account for the contraction bias in a Bayesian framework of decision making. In order to see how the contraction bias emerges from Bayesian inference, we consider a control region in the brain, such as the prefrontal cortex L1 is unknown, L2 is known. Consider a hypothetical subject who forgets the length of L1, but has no ambiguity about the length of L2, i.e., the neural representation of L1 is infinitely noisy whereas there is no noise in the neural representation of L2. In this case, the posterior of L1 is the prior distribution. In contrast, the prior distribution makes no contribution to the posterior of L2. Therefore, the optimal strategy would be to report ‘L1>L2’ in trials where L2 is smaller than the median of the prior distribution and to report ‘L1<L2’ in trials in which L2 is larger than the median. Therefore, in the impossible trials in which L1 = L2, the subject would report ‘L1>L2’ if L1 is smaller than the median of the prior distribution and would report ‘L1<L2’ otherwise, as depicted in L1 and underestimating relatively large L1.L1 and L2 are equally uncertain. Consider a case where the estimated uncertainties in the representations of L1 and L2 are equal. This would be true if the only uncertainty in the representations of L1 and L2 results from sensory noise, and memory storage does not add any additional noise to the representation of L1. In the impossible trials in which the two bars are physically identical, the contribution of the prior distribution to the posteriors of L1 and L2 is equal. Symmetry considerations indicate that the subject would report that ‘L1>L2’ at chance level for all bar lengths, i.e., there is no contraction bias, as depicted in L1 is less certain than L2. In intermediate cases where the level of uncertainty in L1 is larger than that of L2, for example, as a result of added noise due to memory storage, we expect the resultant response curve to reside between the response curves of L1 is reported to be larger than L2 as a function of the lengths of L1 and L2 . Similar to the first experiment, all lengths were presented in logarithmic scale to satisfy the assumption of independence of noise and bar length. We compared the responses of this group to the original group who saw stimuli that were drawn from a uniform distribution in the logarithmic scale between 150 to 600 pixels. The accuracy of subjects in the trials in which L1≠L2  was indistinguishable between the two groups , supporting the assumption that the level of noise in the neural representation of the bars is independent of bar length in these ranges. Response curves for the two groups in the impossible trials are depicted in If the contraction bias results from Bayesian inference, then changing the prior distribution is expected to change the response curve. In particular, assuming that noise is independent of the length of the bars, a translational shift in the prior distribution would result in an equal translational shift in the response curve without changing its shape. To test this prediction, we asked a new group of naïve subjects to participate in the experiment of L1 is 30% higher than the uncertainty in L2 , the intermediate trials in which the difference between L1 and L2 was ±15% (t-test) and the most difficult trials in which the difference between L1 and L2 was ±7.5% (t-test).The distracting task was designed to interfere with the memory of the first bar. In the Bayesian framework, it was intended to add ‘noise’ to the representation of ary task . This dep = 0.0002, two-tailed), supporting the assertion that a decrease in performance is associated with an increase in the magnitude of the contraction bias.In order to characterize the effect of the secondary task on the contraction bias, we compared the response curve of subjects in the impossible trials with interference from the secondary task  the subjects could not know whether they would be presented with an interfering task and therefore could not know what weight to give to the prior distribution. Therefore, if the computation had taken place at the time of the encoding of L1, we would have observed no difference in the slope of the response curve between the two conditions. Therefore, the Bayesian computation necessarily took place after the interfering task, at the time of L1 retrieval or later, when L1 and L2 were compared.Within a single trial, when does information about the prior distribution combine with the sensory measurement? One possibility is that it takes place during the encoding of d trials . In otheHow do subjects learn the prior distribution? In order to address this question, we compared the level of contraction bias, as measured by the slope of the response curve, in the first 20 impossible trials to the slope in the last 20 impossible trials for subjects who completed the experiment in In this study we examined the effect of a translational shift in the prior, but we did not alter the shape of the prior distribution. Previous studies have shown that subjects are sensitive to the shape of the prior distribution in category and sensimotor learning Contraction bias in delayed comparison tasks is a common cognitive illusion observed in many different modalities and under different experimental conditions All subjects gave written informed consent using methods approved by the Massachusetts Institute of Technology Committee on the Use of Humans as Experimental Subjects.Subjects were undergraduate and graduate students from the Massachusetts Institute of Technology. All subjects had normal or corrected-to-normal vision and no subjects took part in more than one of the experiments. Each subject received $10 plus 1 cent for every correct trial in the experiment for a session lasting less than an hour.Stimuli were white horizontal bars on a black background displayed on a 17″ computer screen with a resolution of 1024×768. All bars were 3 pixels wide.L1 at a random location on the screen for 1 sec. After a delay period of 1 sec, during which screen remained blank, L2 appeared at another random location on the screen. L2 remained visible until the subjects pressed one of two keys indicating which bar was longer. The difference in length between 1L and 2L varied between −30% and +30%. Unbeknownst to the subjects, in roughly 50% of the trials, the lengths of the first and second bars were equal (L1 = L2). Subjects did not receive feedback on performance on individual trials. Each trial was followed by a 2 sec intertrial interval during which the screen remained blank. Two distinct groups of subjects completed the standard task. One group (n = 9) saw L1 bars chosen uniformly in the logarithmic scale from the  pixel interval, while the other group (n = 10) saw bars chosen from the  pixel interval.Subjects sat approximately 60 cm from a computer screen in a dimly lit room. Each subject completed 400 to 600 trials in one hour and received feedback on their overall performance after every 20 trials. No other feedback was provided. In the standard task, each trial started with the presentation of a L1 bars were chosen uniformly in the logarithmic scale from the  pixel interval; (2) subjects completed a distracting memory task between the presentation of L1 and L2 in a randomly selected 50% of the trials: 500 msec after L1 disappeared, a random sequence of four colors  were displayed on the screen for 400 msec each. 400 msec after the disappearance of the last color, a number from 1 to 4 appeared in yellow on the screen. Subjects were instructed to recall the color that corresponded to the number and press one of four dedicated keys to indicate this color. L2 appeared 500 msec after subjects made their color choice.The modified task was identical to the standard task with two exceptions: (1) According to our Bayesian hypothesis, the contraction bias emerges because subjects use Bayes' law to combine noisy information about the lengths of the bars with knowledge about the prior information in order to optimize performance. In this section we formalize this intuition.iL and iR be the logarithm of the length of bar i and its neural representation, respectively. We assume that this representation is noisy such that iz is drawn from a zero-mean Gaussian distribution with variance iR for a given representation of bar length In accordance with Weber's law, the lengths of the bars are measured in logarithmic scale. Let  uniform . Bayes' niform  such that L1 has to be stored in memory, a process that may contribute additional noise to the representation of L1. An ideal Bayesian observer, who has access to R1 and R2, would report ‘L1>L2’ in trials in which L1<L2’ in trials in which L1>L2’ in a trial in which L1 and L2 are presented is given byGiven a pair of neural representations, L1 is infinite, whereas there is no uncertainty in the representation of L2. In other words, L1>L2’ if R2 is larger than the median of L1. In trials in which L1 = L2, Eq. (3) dictates that he would report ‘L1>L2’ in trials in which L2 is larger than the median and ‘L1<L2’ otherwise.In order to construct the response curve we compute  . For furAll slopes were computed after normalizing the range of lengths to 0 and 1 in the logarithmic space.We used a pairs bootstrap resampling procedure In order to compare the response curve slopes between subjects who saw 50–200 pixel lines and those who saw 150–600 pixel lines we sampled from each group independently using the algorithm above, and then constructed a 95% confidence interval on the difference between the bootstrap slopes of the two groups.In order to compare trials with and without the interference task we calculated the difference in the bootstrap slope of each subjects' standard and interfered trials, and found the 95% confidence interval of this difference. The same method was also used to compare the slope of the response curve in the first 20 impossible trials of the experiment to the slope of the response curve in the last 20 impossible trials of the experiment.In order to compare behavioral performance to that predicted by the model, we used the model presented above to generate a set of response curves of ideal observers characterized by different values of L2>L1’ in the 50–200 and 150–600 standard experiments. Subjects reported that ‘L1>L2’ in the impossible trials in 41% and 46% respectively. This tendency has been reported previously p = 0.58, two tailed t-test). More importantly, this explanation is circular because a bias in the opposite direction could equally well have been explained by arguing that the prior distribution is biased in favor of large magnitudes. Therefore we did not attempt to account for the global bias and subtracted it before fitting, assuming that it is generated by a different mechanism. Thus, for the purpose of finding the parameters we added a constant to each of the response curves to normalize them such that mean(Pr[‘L1>L2’]) = 0.5. For purposes of comparison, the range of the logarithm of bar lengths was normalized to lie between 0 and 1 and we used a least square fit to find the parameters that best fit the population-average experimental data. We found that the best fit model parameters for the groups who saw 50–200 and 150–600 pixel-long bars were given by Note that subjects exhibited a small bias in favor of reporting ‘


L1 is a cell adhesion molecule associated with a spectrum of human neurological diseases, the most well-known being X-linked hydrocephalus. L1 knockout (L1-KO) mice have revealed a variety of functions of L1 that were crucial in brain development in different brain regions. However; the function of L1 in neuronal migration during cortical histogenesis remains to be clarified. We therefore investigated the corticogenesis of mouse embryos in which L1 molecules were knocked down in selected neurons, by employing in utero electroporation with shRNAs targeting L1 (L1 shRNA). Although more than 50% of the cells transfected with no small hairpin RNA  vector at embryonic day 13 (E13) reached the cortical plate at E16, significantly fewer (27%) cells transfected with L1 shRNA migrated to the same extent. At E17, 22% of cells transfected with the MGFP-only vector were found in the intermediate zone, and significantly more (34%) cells transfected with L1 shRNA remained in the same zone. Furthermore, the directions of the leading process of neurons transfected with L1 shRNA became more dispersed compared with cells with the MGFP-only vector. In addition, two transcription factors expressed in the neurons, Satb2 and Tbr1, were shown to be reduced or aberrantly expressed in neurons transfected with L1 shRNA. These observations suggest that L1 plays an important role in regulating the locomotion and orientation of migrating neurons and the expression of transcription factors during neocortical development that might partially be responsible for the abnormal tract formation seen in L1-KO mice. © 2012 Wiley Periodicals, Inc. The neural cell adhesion molecule L1 is one of the most intensively studied adhesion molecules expressed in the developing central and peripheral nervous systems , agenesis/dysgenesis of the corpus callosum, and X-linked spastic paraplegia and CRASH syndrome  strategy and in utero electroporation  was used in this study. The small interfering RNA (siRNA) sequence targeting L1 was designed and provided by SuperArray Bioscience Corporation . We prepared four shRNAs targeting L1 cloned in pGeneClip hMGFP vector and a negative control shRNA (shNC) showing no homology to any of the known mammalian genes , as well as hMGFP-labeled mock plasmid (MGFP only plasmid). The siRNA sequences are shown in 6 dissociated cells were suspended in 100 μl of transfection reagent, and the cells were then transfected with 10 μg of shRNA using the Amaxa Nucleofector , according to the manufacturer's instructions, using program No. O-005. We incubated the shRNA-transfected cells in 5 ml Neurobasal medium (Invitrogen Japan), supplemented with 2% B27 , 0.5 mM L-glutamine , and 1% penicillin/streptomycin solution . We added 4 ml of 1 × 106/ml untransfected cells into the 1 ml shRNA-transfected cells (at a density of 1 × 106/ml) and plated mixed cells at a final density of 1 × 106 cells/ml in 24-well plates, which had been precoated with 0.1 mg/ml poly-D-lysine (PDL) and 50 μg/ml laminin (Invitrogen Japan) or 0.1 mg/ml PDL, 40 μg/ml anti-Fc antibody, and 1 μg/ml L1-Fc. All of the cultures were maintained at 37°C in a 95% air, 5% CO2 (v/v) humidified atmosphere. All animal studies were approved by the Institutional Review Board for Biomedical Research using Laboratory Animals at Kyoto Prefectural University of Medicine, and the animals were handled according to the institutional guidelines and regulations.Dissociated cultures of cortical neurons were prepared from embryonic day 13.5 murine fetus . Briefly, the dorsal forebrain was dissected and treated in 0.125% trypsin/0.5 mM EDTA for 15 min at 37°C and then dissociated by gentle trituration (in a Pasteur pipette). In total 5 × 10Gapdh, Rplp1, and Rsp18.Total RNA was extracted from the transfected cultured cells using an RNeasy Micro Kit (Qiagen Japan), and cDNAs were synthesized and amplified as described previously. Briefly, first-strand cDNAs were synthesized using an iScript cDNA Synthesis Kit (Bio-Rad Japan) with oligo(dT) primer. The relative gene expression levels of L1 were measured by qRT-PCR on an ABI Prism 7000 sequence detection system (Applied Biosystems Japan) using SYBR Premix Ex Taq  according to the manufacturer's protocol. The optimized number for thermal cycling was set at 40. The primers for L1 were designed as follows: L1cam_2: 5′-gctttgcctccgagggctgg-3′ (forward), 5′-ccagggacctgtactcgccga-3′ (reverse) and L1cam_6: 5′-ccaagtggagttccgctggacg-3′ (forward) and 5′-cttcggccacttgggggcac-3′ (reverse). TaqMan rodent control reagent (Applied Biosystems Japan) was employed for housekeeping genes, such as P < 0.05 was designated as statistically significant.To evaluate the functional loss of L1 by shRNA targeting L1, at 48 or 96 hr after transfection, the primary cortical neurons were fixed with 4% paraformaldehyde for 5 min at 4°C. After fixation, we observed electroporated neurons expressing MGFP under a confocal microscope . The length of the longest neurite, the number of branches, and the total length of the neurites were measured in NeuronJ, which is an ImageJ plugin allowing the tracing and quantification of elongated structures. Because the shRNA2-transfected cells showed a unique histogram with regard to the longest neurite extension at 48 hr after transfection, we classified the cells into three types, according to the longest neurite length: 1) neuron type 1, longest neurite less than 80 μm; 2) neuron type 2, longest neurite between 80 and 220 μm; and 3) neuron type 3, longest neurite more than 220 μm. All values are expressed as means ± SEM. The SEM values indicate the variation between mean values obtained from at least three independent experiments. Statistical comparisons were conducted by using Dunnett's multiple comparison test. C57BL/6J pregnant mice were purchased from CLEA Japan Inc. . The day on which the vaginal plug was found was designated as embryonic day 0 (E0). In utero electroporation was performed as described previously . After blocking with 10% goat serum, 1% bovine serum albumin (BSA), 0.01% Triton X-100 (Sigma-Aldrich Japan) in 0.1 M PBS, the sections were incubated in primary antibody at 4°C overnight; doublecortin , Satb2 , Ctip2 , or Tbr1 , diluted in 0.1% Triton X-100 and PBS. The sections were rinsed several times with PBS and then incubated in the secondary antibody . The sections were then coverslipped and observed under a confocal microscope .t-test was performed to detect the differences in the dispersion of the “cell process angle.”Images acquired with the Olympus FluoView FV1000 confocal microscope were processed with an Olympus FV10-ASW 1.7 viewer to adjust color and contrast. To conduct a quantitative analyses of the transfected cells, which were clearly detectable with MGFP fluorescence, at least three independent brains were analyzed at each embryonic stage: transfected at E13 and analyzed at E16 or transfected at E13 and analyzed at E17. The wall of the embryonic dorsal brain was divided into six layers, the ventricular/subventricular zone (VZ); the upper and lower intermediate zone (IZ); and the upper, middle, and lower cortical plate CP; D. The waP < 0.05 by one-way ANOVA; The expression level of L1 mRNA was significantly reduced in transfected cortical cells with shRNA2 plasmids at 48 hr after transfection compared with the negative control plasmid shRNA (shNC), no shRNA (MGFP only), as well as with no treatment , and MGFP-only plasmid by in utero electroporation at embryonic day 13 (E13). In utero electroporation allowed us to introduce shRNA into the fetal VZ to knock-down the targeted gene so that neuronal migration from the VZ toward the CP could be traced. We examined the distribution of MGFP-labeled cells in the presumptive somatosensory cortical area of the dorsolateral forebrain, which was located at the level of the medial and lateral ganglionic eminences by coronal section . MGFP-onWhen we observed the migration pattern of the GFP-expressing cells in the fetal telencephalon, we found that the cells transfected with MGFP-only plasmid showed a fine neurite fasciculation and radial migration to the marginal zone, similar to that found in the physiological situation. However, the neurons transfected with shRNA2 showed a wide variation in the orientation of neurites during migration. To confirm the difference in terms of the direction of cellular migration, we measured the angle of the leading neurite relative to the orientation of the CP, as described in Materials and Methods A,B. StatTo investigate whether the process of neuronal differentiation is affected by L1 knockdown, we studied the expression patterns of the transcription factors that are known to regulate the differentiation of cortical neurons, such as Satb2, Ctip2, and Tbr1. These factors are all expressed specifically and associated with the cortical layering. Satb2  belongs to the family of DNA-binding proteins, which regulates gene expression through chromatin modification and interacts with other proteins. It is expressed in corticocortical projection neurons. Ctip2, belonging to the family of zinc finger transcription factors, is expressed in subcortical projection neurons (Leid et al.,Nontransfected neurons observed in both MGFP-only plasmid- and shRNA2-transfected dorsal telencephalon at E16 or E17 showed a similar immunoreactivity for these transcription factors. Satb2 expression was found from the upper to the middle portion of the CP and was expressed more weakly in the IZ. Many MGFP-only plasmid-transfected neurons migrated into the upper and middle CP, showing intense immunoreactivity for Satb2 at E16 as well as at E17, whereas shRNA2-tranfected neurons showed immunoreactivity for Satb2 mainly in the subplate and IZ at E16 and E17 arrow, . Ctip2 iWe found that the downregulation of L1 using in utero electroporation of shRNA affected the radial migration of cortical neurons, with aberrant expression of the transcription factors that are important for cortical layer formation. L1 plays a variety of functional roles required for normal brain development in different brain regions (Lemmon et al.,Our observations suggested that downregulation of L1 induced either a delay in the exit of neurons from the VZ to the IZ or an inability to sense a molecular cue for neurons to migrate into the CP. Our study showed an increased ratio of multipolar shRNA2-tranfected neurons stuck in the IZ at E17, which partially resembled the migration disorder observed in doublecortin loss-of-function mice (LoTurco and Bai,We have demonstrated that downregulation of L1 perturbed not only the locomotion of neurons but also the orientation of the leading process. It is known that the intracellular domain of L1 contains an ankyrin-bindng region and a neuron-specific sequence, RSLE, which is critical for sorting L1 to axonal growth cones (Kamiguchi and Lemmon,To assess this phenomenon followed by downregulation of L1 and disturbed neuronal migration, we examined how the expression of transcription factors known to be involved in cortical development is affected. Satb2 is expressed in corticocortical projection neurons and plays essential roles in the axonal pathfinding of projection neurons in brain development (Britanova et al.,In conclusion, we have demonstrated that downregulation of L1 by using in utero electroporation of shRNA perturbed the locomotion and orientation of radially migrating neurons in neocortical histogenesis, together with alterations in the expression of transcription factors involved in cortical development. These results first indicate that L1 plays an important role not only in axonal guidance but also in neuronal migration during neocortical development. The methodological advantages of the in utero electroporation method we employed allowed us to downregulate L1 in only a subset of cortical cells, so that the novel functions of L1 in cortical development could be elucidated.


It is found that graphene layers can be joined by different domains, with a relative misorientation of 30°. These graphene layers can be decoupled from Cu(110) through low temperature thermal cycling.Revealing the graphene growth mechanism at the atomic-scale is of great importance for achieving high quality graphene. However, the lack of direct experimental observation and density functional theory (DFT) verification hinders a comprehensive understanding of the structure of the carbon clusters and evolution of the graphene growth on surface. Here, we report an in-situ low-temperature scanning tunneling microscopy (LT-STM) study of the elementary process of chemical vapor deposition (CVD) graphene growth via thermal decomposition of methane on Cu(110), including the formation of monodispersed carbon clusters at the initial stage, the graphene nucleation and the ripening of graphene islands to form continuous graphene film. STM measurement, supported by DFT calculations, suggests that the carbon clusters on the surface are C Ever since its mechanical exfoliation from small mesas of highly oriented pyrolytic graphite, graphene has spurred a tremendous of interest because of its exceptional electronic and mechanical properties, such as anomalous quantum Hall Effect (QHE), long-range ballistic transport, high carrier mobility, tunable band gap, high elasticity and intrinsic strength1231291011141516171819202122232425The graphene growth on TM surfaces is based on high-temperature pyrolysis of hydrocarbons and different growth mechanisms can be involved according to the carbon solubility limit in the metal. For the growth on TM where carbon is soluble, the graphene forms when the sample is cooled and carbon segregates on the surface (surface segregation)2930312H2 can be easily formed on a Cu(111) surface, which represents a more favorable reaction path compared to CH dissociationN on Ni(111), Gao et al. indicated a ground state structure transition from a one-dimensional (1D) carbon chain to a two-dimensional (2D) sp2 carbon network at N ~ 10–1221 is a very stable magic carbon cluster on Rh(111), Ru(0001), Ni(111) and Cu(111) surfaces1516Graphene growth has been demonstrated on a variety of TMs. For example, Li and colleagues reported a CVD method that used copper-foil to produce single crystal graphene with dimensions of up to 0.5 mm14333435363738394041424) on Cu(110) using low-temperature scanning tunneling microscopy (LT-STM), including the formation of monodispersed carbon clusters at low temperature, nucleation and ripening of graphene islands at high temperature. Combined with first principles calculations, the monodispersed carbon clusters are identified as C2H5. Different domains stitch together to form a graphene layer, with a preference angle of 30° at the grain boundaries. These graphene layers can be decoupled from Cu(110) through low temperature thermal cycling.Here, we report an atomic scale characterization of the elementary process of CVD graphene growth via thermal decomposition of methane (CH4 at room temperature (RT) and subsequent annealing at 480°C in CH4 at a pressure of 2 × 10−5 mbar for 50 min, the Cu(110) surface was almost decorated with carbon clusters of monodispersed size. Each carbon cluster appears as a bright spot with an identical size of 0.4 nm. Careful inspection of the STM image reveals that the surface is decorated by isolated but well-defined superstructures, where the carbon clusters are adsorbed in an epitaxial relationship with the underling Cu(110). As indicated by the dashed lines in 0 of 0.512 nm; while it is 2b0 of 0.723 nm between two columns (a0 and b0 are the unit cell dimensions of Cu(110)). It can also be revealed that the carbon cluster arrays are aligned precisely with the crystal orientation of the underlying Cu(110). The carbon cluster at this low coverage was referred to as “cluster 1” with a density around 2.70 × 1014/cm2. Previous theoretical studies proposed that carbon dimmers are energetically favorable on the Cu surface37382Hx).As shown by the high magnification STM image in 14/cm2, referred to as “cluster 2”. In this regime, the arrangement is supposed to be cluster-cluster interaction dominated. Some brighter lines can be frequently observed, induced by the stress relaxation at high cluster coverage with increased lateral inter-cluster interaction.Further increasing the coverage of the carbon clusters can result in the formation of a hexagonally close packed structure, as shown in 4 at a pressure of 2 × 10−5 mbar for 130 min can promote the nucleation of small graphene flakes. As shown in Annealing the Cu(110) surface at high temperature at 550°C in CH1Hx (0 ~ 4) and C2Hx (0 ~ 6) clusters on Cu (110) were studied. We define the formation energy in tot is the total energy of the adsorbed system, Esub is the energy of clean Cu (110) substrate, μi and ni  represent chemical potential and the number of atoms in the cluster, respectively. Considering the equilibrium of CH4 and H2, the relationship of μH and μC in unit of electron volt can be obtained as 4 and H2.To obtain the atomic structure of the carbon clusters, the adsorption of various carbon clusters on Cu(110) were simulated using DFT. First, the stability of Clong site), bridge-short site (Bshort site) and Top site (T-site)2). The χ here was set to be 20:1; we also tested χ = 1:20, which gave similar results.For each carbon cluster species, the most stable adsorption configuration was found by checking different adsorption sites on Cu(110) surface, including the hollow site (H-site), bridge-long site  at high temperature. The average lifetime of C2H6 and C2H5 can be estimated by their adsorption energy Ea via 2H6 and C2H5 on Cu(110) surface are 0.41 and 2.85 eV, respectively. υ0 is about 1013 s−1. Therefore, their average lifetime on the surface at 480°C is 5.5 × 10−11 and 1.2 × 106 s, respectively. Such a short lifetime makes C2H6 not be able to be observed by STM. Therefore, C2H5 could be the most possible abundant species from the thermodynamic point of view.From 2, C2H4, C2H5 and C2H6. The optimized unit cell of the carbon cluster is 2a0 = 0.504 nm, 2b0 = 0.713 nm. Among these carbon clusters, the simulated STM image of C2H5 is in good agreement with the experimental results. All other stable species cannot reproduce the experimental circular shape. Hence, the basic structures of the carbon clusters are elucidated by the STM images in combination with DFT calculations as C2H5.STM images of several partially dehydrogenated carbon dimer species were also simulated using the Tersoff and Hamann approximationLarge graphene flakes can be achieved through low temperature thermal cycling process as described in the 2519As described in the 4 decomposition, similar to the previously reported hydrogenThe formation of such physically decoupled graphene can arise from the intercalation at the graphene/Cu(110) interface by hydrogen atoms released from CH4. Low temperature annealing (>480°C) in CH4 results in the formation of carbon clusters at the initial stage; further high temperature annealing (>550°C) activates the graphene nucleation; prolonged annealing in the absence of CH4 propels the diffusing and ripening of these graphene island to form continuous graphene films extended over the surface. Low temperature thermal cycling induced decoupling of graphene from Cu(110) has also been demonstrated. Our systematic investigations identify the fundamental carbidic building blocks by STM measurement, and further elucidate their atomic structures through DFT calculations. Our work could lay the foundation for providing rational design rules for synthesis of large area single crystalline graphene films.Through the combination of the LT-STM and DFT calculations, we reveal the elementary process of graphene growth on Cu(110) surface via thermal decomposition of CH4. Prior to the deposition of CH4, Cu(110) substrate was cleaned by a few cycles of Ar+ ion bombardment and subsequent annealing at 530°C. The CH4 gas was introduced into the growth chamber through a leak valve, and the pressure was monitored by a cold cathode gauge. A typical growth procedure is as follows: the Cu(110) substrate was exposed to CH4 at a pressure of 2 × 10−5 mbar for 20 min; annealing the sample at 480°C in CH4 at a pressure of 2 × 10−5 mbar resulted in the formation of carbon clusters; further annealing the sample in the absence of CH4 at 550°C initiated the graphene nucleation; prolonged annealing without CH4 at higher temperature up to 720°C propelled the ripening of graphene islands.Graphene was grown on a single crystal Cu(110) via thermal decomposition of CH−10 mbar, housing an omicron LT-STM interfaced to a Nanonis controller. All STM imaging were performed at 77 K using constant current mode with an electrochemically etched tungsten tip. All the bias voltage was applied to the tipThe LT-STM experiments were carried out in a custom-built multichamber ultra-high-vacuum (UHV) system with base pressure better than 1.0 × 101Hx (0 ~ 4) or C2Hx (0 ~ 6) clusters on Cu (110) surface were studied using DFT calculations. A 5-layer slab with a 20 Å vacuum layer was used as the substrate. The bottom layer was fixed to its bulk configuration and all other atoms were fully relaxed. A (3 × 4) supercell was chosen to make sure that clusters were separated to their neighboring clusters by more than 10 Å. In STM simulation, a (2 × 2) supercell was chosen according to the experimental coverage.Stability of C−5 eV and 0.02 eV/Å. For the (3 × 4) and (2 × 2) models, (7 × 7 × 1) and (10 × 14 × 1) k-point grids were used, respectively. STM images were simulated using the Tersoff and Hamann approximationAll the calculations were performed using DFT implemented in the Vienna Ab Initio Simulation Package (VASP) within the generalized gradient approximation55W.C. conceived and designed the experiments; J.L.Z. and T.C.N. performed the experiments; Z.Z.W., S.N.W. and Z.Y.L. performed theoretical calculations; W.C. and J.L.Z. wrote the manuscript. All authors contributed to writing and revising the manuscript.Supplementary Information


A simple two-step method was employed for preparing nano-sized gold nanoparticles-graphene composite to construct a GNPs-GR-SDS modified electrode. Hemoglobin (Hb) was successfully immobilized on the surface of a basal plane graphite (BPG) electrode through a simple dropping technique. Direct electrochemistry and electrocatalysis of the hemoglobin-modified electrode was investigated. The as-prepared composites showed an obvious promotion of the direct electro-transfer between hemoglobin and the electrode. A couple of well-defined and quasi-reversible Hb CV peaks can be observed in a phosphate buffer solution (pH 7.0). The separation of anodic and cathodic peak potentials is 81 mV, indicating a fast electron transfer reaction. The experimental results also clarified that the immobilized Hb retained its biological activity for the catalysis toward NO. The biosensor showed high sensitivity and fast response upon the addition of NO, under the conditions of pH 7.0, potential -0.82 V. The time to reach the stable-state current was less than 3 s, and the linear response range of NO was 0.72-7.92 μM, with a correlation coefficient of 0.9991. Due t in 2004 . Like ca carbon)  that adh carbon) –10.et al. [2O2. Huang et al. [2 nanocomposite could effectively facilitate the direct electron transfer of catalase to the electrode.On the other hand, due to its low-cost, large specific surface area and strong interactions with metal clusters, graphene is a promising material for catalytic applications . The diset al.  employedg et al.  also shoHowever, to date, there are few papers concerning the electrochemical response of hemoglobin on gold nanoparticles-graphene sheets composites. As a well-characterized, electron transfer protein, hemoglobin has been used extensively as a test system for direct electron transfer (DET) of redox proteins or for communication in a protein stack by assembling it with other proteins in a matrix –25.In this work, we synthesized Au nanoparticles using graphene oxide (GO) as protecting agent to give a stable red suspension. To obtain high electrical conductivity and water solubility of our GNPs-graphene composites, we added sodium dodecyl sulfate (SDS) solution to the GNPs-GO suspension prior to chemical reduction. The anionic surfactant SDS was employed as an effective solubilizing agent for graphene nanosheets and for isolating them from each other. Otherwise, the strong van der Waals interactions among the reduced graphene sheets results in their rapid aggregation in solution during the chemically reduction process. Then, water soluble GNPs-GR can be successfully obtained by the non-covalent interaction of graphene with sodium dodecylsulphate (SDS) in this procedure ,27. Furt2.2.1.Electrochemical experiments were performed with a CHI660 electrochemical analyzer  with a conventional three-electrode cell. A Hb/GNPs-GR-SDS modified BPGelectrode (Φ = 5.2 mm) was used as a working electrode. An Ag/AgCl electrode and a platinum wire electrode were used as the reference and the auxiliary electrodes, respectively.−1) was stored at a temperature of 4 °C as stock solution. Graphite, and hydrazine hydrate solution (50 wt %) were purchased from Shanghai Chemical Reagent Co. Ltd.  Saturated NO solutions were prepared as described in the previous literature [Horse heart hemoglobin was obtained from Sigma  and used without purification. Hb solution (5 mg·mLterature . In brie2.2.4 with NaBH4 in the presence of aqueous graphene oxide (GO). An aqueous solution of HAuCl4  was added into graphene oxide aqueous solution  and stirred for 5 min. After that, slow and controlled addition of NaBH4 solution  is essential to avoid gold nanoparticles from forming too fast and aggregating. This experiment was repeated with graphene oxide concentrations of 0.02, 0.11 and 0.27 mg· mL−1 as well as one experiment where no graphene oxide was added.The oxidation of the graphite was carried out based on the Hummers method . The obt2.3.SDS solution  was added to the obtained gold nanoparticles-graphene oxide suspension and stirred for 20 min. Then, hydrazine hydrate solution (20 μL) was added and stirred for 5 min. This mixture was reacted at 100 °C for 24 h. The GNPs-GO solution changed color, from red to black, after the hydrazine-vapor treatment, indicating reduction of the graphene oxide . The res2.4.−1 Hb for 3 h to form an Hb/GNPs-GR-SDS/BPG electrode. Prior to electro-chemical experiments, the electrode was rinsed thoroughly with doubly distilled water andstored at 4 ° C. The Hb/GR-SDS modified electrode without GNPs and the Hb/GNPs-SDS modified electrode without graphene were prepared in the same way, only omitting the steps of the addition of HAuCl4 solution and graphene oxide solution, respectively.Initially, the BPG electrode was polished with abrasive paper before each experiment. After a short rinse with distilled water and ultrasonic cleaning with distilled water and ethanol, the electrode was allowed to dry at room temperature. Then, the GNPs-GR-SDS suspension (10 μL) was cast on the surface of a pretreated BPG electrode with a microsyringe and the solvent (water) was allowed to evaporate at ambient temperatures. The electrode was denoted as the GNPs-GR-SDS/BPG electrode. Finally, this GNPs-GR-SDS/BPG electrode was immersed in a phosphate buffer solution (pH 7.0) containing 5 mg·mL3.3.1.−1 graphene oxide was added into the solution, the color of the solution turned a red wine color. Reducing the amount of graphene oxide gradually, the color of the solution turns red, then purple, blue, which could be explained by the aggregation of gold nanoparticles. At a low graphene concentration (0.02 mg·mL−1), the gold particles are suspended but in an aggregated state. Without the addition of any dispersant, the suspension flocculates very rapidly, which should indicate a strong agglomeration of the nanoparticles.A photograph of five GNPs-GO suspensions with different graphene oxide concentrations is shown in max from 520 to 556 nm, which is typical for increasingly larger GNP diameters. Similar red-shifts have been observed in the measured optical spectra of gold nanoparticles and are attributed to the effect of electromagnetic retardation in larger nanoparticles [articles ,31.In other words, gold nanoparticles-graphene oxide composites were successfully synthesized using graphene oxide as a dispersing agent to optimize the dispersion and the stability of the gold nanoparticles. Unlike the methods reported in previous literatures, the present method involves a facile fabrication route for the attachment of gold nanoparticles on the surface of graphene without anyorganic reagent. The resulting GNPs-GO suspension can remained as a fine dispersion at least two months. Subsequently, we added hydrazine hydrate to the GNPs-GO suspension to obtain a gold nanoparticle-graphene solution in the presence of sodium dodecyl sulfate.−1 graphene. The SEM analysis indicates that the gold nanoparticles are uniformly deposited on the graphene surface, with an average size of 50 nm. This fact showed that the presence of the oxygen functionalities at the graphene surface may play an important role on the nucleation and growth of gold nanoparticles. Furthermore, 3.2.2-saturated Hb-free phosphate buffer solution (pH 7.0). The Hb/GNPs-GR-SDS/BPG electrode gives a couple of stable and well-defined redox peaks, where anodic and cathodic peak potentials are located at −317 and −398 mV, respectively  can be estimated according to Laviron [−1. This value of Ks of our prepared modified electrodes is higher than Hb/graphene-Pt/GCE (0.14 s−1) [ Laviron , which i.14 s−1) . This in−1, less than the theoretical value of -59 mV pH−1 at 25 °C for a reversible proton-coupled single electron transfer [The effect of the pH of the supporting electrolyte on the peak potentials of the Hb was also investigated . Both retransfer ,36. Althtransfer  and Hb-Dtransfer  films:H3.3.The resulting Hb/GNPs-GR-SDS/BPG electrode exhibited a good electrocatalytic activity toward the reduction of NO. To further examine the response feature of the Hb/GNPs-GR-SDS electrode to NO, the amperometric response of the Hb/GNPs-GR-SDS modified BPG electrode to NO was recorded while successively adding NO to a continuous stirred PBS solution. In this process, a potential of −0.82 V was applied to the working electrode .−8 mol·L−1 based on 3 signal-noise ratio. The resulting response suggests that the Hb/GNPs-GR-SDS/BPG electrode can be used as a reagentless biosensor to detect NO. The determined parameters of this sensor and other modified electrodes used for the determination of NO are listed in The reductive current increases to reach a stable plateau within 3 s when NO is added to the buffer solution. This suggested that the response of the electrode to NO should be a quick responsive process. The experimental results showed that the amperometric response current is linear when the concentration of NO is in the 0.72-7.92 μM range. In addition, the detection limit of the biosensor was evaluated to be 1.2 × 103.4.The stability of the Hb/GNPs-GR-SDS electrode was investigated. Even when 400 continuous cyclic scans were carried out in the potential range from 0.1 V to −1.0 V with a 100 mV/s scan rate, no obvious change of the CV curve can be observed, suggesting that proteins can be tightly immobilized on the surface-modified BPG electrode. Eight repetitive experiments were carried out with the buffer solution containing 0.72 μM NO. The relative standard derivation for eight repetitive measurements isbelow 2.3%. The Hb/GNPs-GR-SDS modified electrode that was left to stand in pH 7.0 PBS could keep its activity for at least two weeks.4.In conclusion, a gold nanoparticle-graphene nanosheets suspension can be prepared though the chemical reduction method using SDS as protector and disperser. Hb adsorbed on the GNPs-GR-SDS modified electrode via the electrostatic interactions retains its native secondary protein structure and the direct electron transfer was achieved. The CVs of a Hb/GNPs-GR-SDS/BPG modified electrode exhibited a pair of redox peaks corresponding to the electroactive center of Hb and showed good catalytic activity for the reduction of NO. The developed approach can be potentially useful in the fields of catalysis, fuel cells, and other related fields.


The transactive response DNA-binding protein (TDP-43) is a major component of the abnormal intracellular inclusions that occur in two common neurodegenerative diseases of humans: (1) a subtype of frontotemporal lobar degeneration and (2) amyotrophic lateral sclerosis. Genetics, experiments in cultured cells and animals, and analogy with other neurodegenerative diseases indicate that the process of TDP-43 aggregation is fundamental to the pathogenesis of these 2 diseases, but the process by which this aggregation occurs is not understood. Biochemical fractionation has revealed truncated, phosphorylated and ubiquitinated forms of TDP-43 in a detergent-insoluble fraction from diseased CNS tissue, while these forms are absent from controls. However, a large amount of the normally predominant 43-kDa form of TDP-43 is present in the detergent-insoluble fraction even from control brains, so it has not been possible to determine if this form of TDP-43 is part of pathological aggregates in frontotemporal lobe degeneration. We used semi-denaturing detergent-agarose gel electrophoresis to isolate high molecular weight aggregates containing TDP-43 that are present in the cerebral cortex of individuals with frontotemporal lobar degeneration but not that of controls. These aggregates include the same covalently modified forms of TDP-43 seen in detergent-insoluble extracts. In addition, aggregates include a 43-kDa TDP-43 species. This aggregated 43-kDa form of TDP-43 is absent or present only at low levels in controls. The presence of 43-kDa TDP-43 in aggregates raises the possibility that covalent modification is not a primary step in the pathogenic aggregation of TDP-43 associated with frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Frontotemporal lobar degeneration (FTLD) is a dementia syndrome defined by both clinical and pathological features. The prevalence of FTLD is approximately 15/100,000 persons aged 45–65 years TARDBP gene, which encodes TDP-43, are linked to rare familial forms of ALS Remarkably, abnormal intracellular accumulations of TDP-43 are also prominent in most cases of amyotrophic lateral sclerosis (ALS), a condition in which motor neurons of both the brain and the spinal cord degenerate, causing progressive weakness of skeletal muscle Serial extraction of brain homogenate in buffers of increasing stringency yields a fraction that is insoluble in a solution of 500 mM NaCl and 1% N-lauroylsarcosine Problematically, the detergent-insoluble fraction from both FTLD-TDP and control brains harbors a large amount of 43-kDa TDP-43 To better characterize the TDP-43 species in FTLD-associated aggregates, we employed an alternative method to detergent extraction, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE), to isolate high-molecular-weight complexes containing TDP-43. We then analyzed the components of these complexes by SDS-PAGE and immunoblotting. In addition to covalently modified forms, described above, we found elevated levels of a 43-kDa TDP-43 species in the FTLD-TDP aggregates.SDD-AGE has been described as a means of revealing the aggregated proteins known as “yeast prions” In some control cases a “tail” of TDP-43 signal extended into the upper regions of the gel. To objectively confirm the difference in TDP-43 migration between cases and controls, we compared the ratio of the relative quantity of TDP-43 in a segment extending from 5 mm to 30 mm below the well (encompassing the densest portion of the broad high molecular weight band), to the relative quantity in a 20 mm segment encompassing the TDP-43 signal in the lower portion of the lane that was present in all samples. This ratio was greater in every FTLD-TDP case than in any control , and theThe FTLD-associated high molecular weight band was present when immunoblots were probed with either a polyclonal antiserum or an independently derived monoclonal antibody to TDP-43, but not when blots were probed with the secondary antibody alone . SimilarWe devised methods to investigate which TDP-43 species contribute to the aggregates isolated by SDD-AGE. First, we used large format gels to increase the amount of brain homogenate we could separate by SDD-AGE . We thenTo estimate the overall recovery efficiency of this method, we loaded pre-stained molecular weight standards  on a large format SDD-AGE gel. Unlike brain homogenates, all proteins from the pre-stained marker could be observed to run in a relatively small region toward the bottom of the SDD-AGE gel. We excised the markers in a 3.4 cm long fraction of the lane and concentrated the markers using the method described above. A sample from the final 2 mm of the concentrating column and dilutions of pre-stained standards were compared on SDS-PAGE gels. Approximately 91±11 (s.d.)% of the marker was recovered in the final 2 mm section. Since 85% of the total detectable brain protein was found in the final 1.5 mm of the column, and approximately 90% of total protein was recovered from the final 2 mm, we conclude this method efficiently concentrates approximately 77% of the total protein in an excised SDD-AGE gel region into the final 1.5 mm of the column.We compared proteins concentrated from the aggregate and monomer regions on silver stained SDS-PAGE gels. The aggregate region contains a complex mixture, with at least 40 individual protein bands revealed by silver stain . The silHaving developed a method to efficiently concentrate proteins from excised regions of SDD-AGE gels, we proceeded to analyze the TDP-43 components of the aggregate region. SDS-PAGE immunoblots of concentrates from the aggregate region revealed several species of TDP-43 . All FTLConcentrates from the “monomer” region revealed a 43-kDa form of TDP-43 as the major species in both control and FTLD brain homogenates. Despite the apparently similar amounts of TDP-43 in the “aggregate and “monomer” regions in images of SDD-AGE immunoblots of FTLD brains, the amount to TDP-43 in the monomer region seemed to be much greater than in the aggregate region (data not shown). Precise quantitation was not possible because of the differing distributions of the TDP-43 species on PAGE immunoblots between the aggregate monomer regions.Immunoblots probed with a monoclonal antibody recognizing TPD-43 phosphorylated at serines 409 and 410 (pS409/410) reveal that most of the TDP-43 bands from FTLD-TDP aggregates contain phosphorylated TDP-43 species . HoweverWe considered whether endogenous phosphatase activity might have generated the 43-kDa TDP-43 species from a full-length phosphorylated species, such as that composing the 45-kDa band. To determine whether such activity was present during our aggregate preparation procedure, brain homogenates were prepared from frozen tissue under conditions designed to either minimize or enhance the activity of hypothetical phosphatases, then concentrates of the aggregate portion of these homogenates prepared. We predicted that if phosphatase activity were present in the homogenates, then the ratio of the 43-kDa species to the 45-kDa species would increase under the enhancing conditions. No significant effect was observed .SDD-AGE separates aggregated TDP-43 species that are present in FTLD-TDP brain from forms of TDP-43 that are found in both FTLD-TDP brains and controls. SDD-AGE is thus a simple and rapid assay for aggregated TDP-43 that may prove useful in future studies of the nature and distribution of these pathological aggregates.In SDD-AGE gels, the predominant lower molecular weight species in both controls and FTLD cases migrates with an apparent molecular weight of about 170 kDa, whereas is SDS-PAGE gels the normally predominant band migrates as a 43-kDa band. Since SDD-AGE only partially denatures proteins, migration rates would not be expected to be directly proportional to molecular mass. The apparent larger size of the predominant TDP-43 species in SDD-AGE gels may reflect slowed migration of this species in a partially unfolded state or interactions of the migrating TDP-43 peptide with other species.Since the aggregates seen with SDD-AGE are present only in FTLD-TDP cases, all TDP-43 species migrating in these aggregates are likely to be disease-related. In contrast, the TDP-43 species isolated by the serial extraction method that is commonly used to enrich for FTLD-associated forms of TDP-43 also include a substantial amount of TDP-43 that is not specifically associated with disease TDP-43 aggregates are found in a region of SDD-AGE gels that contain only about 1% of the total protein in the lane. A number of proteins other than TDP-43 were isolated from this region in both FTLD cases and controls. Perhaps surprisingly, the TDP-43 bands revealed by immunoblots of protein from this region are not distinctly visible in silver-stained gels. This may reflect the low total amount of TDP-43 protein in this region, the diffuse nature of most TDP-43 bands or a lack of affinity of TDP-43 species for the silver stain. Whether other proteins from the aggregate region are also aggregated, whether they are in the same cellular compartment as aggregated TDP-43, and whether other proteins from this region are specifically found in FTLD is not known, but these are potentially fruitful areas for future studies.The TDP-43 species extracted from the aggregates in SDD-AGE gels appear to include all the modified forms of TDP-43 forms that had been previously identified in the detergent-insoluble fraction from patients with FTLD-TDP Our most significant finding, however, is that a 43-kDa species of TDP-43 is present in FTLD-associated TDP-43 aggregates. The aggregated 43-kDa species migrates in SDS-PAGE identically to the physiologically predominant TDP-43 species, and lacks obvious covalent modifications. Like the physiologically predominant species, it is not phosphorylated at the 409/410 sites. Known sites of TDP-43 phosphorylation in FTLD include serines at codons 379, 403, 404, 409 and 410 This aggregated 43-kDa species is present at significantly higher levels in FTLD-TDP cases than in controls. To our knowledge, these studies represent the first demonstration of aggregated 43-kDa TDP-43 in diseased human brain. Immunohistochemical studies had indicated the presence of “full length” TDP-43 in intracellular inclusion in both FTLD-TDP and ALS, but it is not possible from such studies to know if the full-length TDP-43 had undergone phosphorylation or other covalent addition of a chemical group The forms of TDP-43 found in disease-associated aggregates may provide insight into the process of pathological aggregation in FTLD. To summarize, these species include at least 2 forms whose polypeptide length appears to be the same as the physiologically predominant 43-kDa species (a form not phosphorylated at S409/410 and a phosphorylated form); several truncated forms, at least some of which are phosphorylated; and forms with molecular weights greater than 45 kDa that are phosphorylated. Species other than the 43-kDa and 45-kDa forms may be ubiquitinated as well. .in vivo or in the interval between death and autopsy. Excepting this possibility, the presence of TDP-43 without apparent phosphorylation, truncation or ubiquitination in aggregates indicates that none of these covalent modifications is necessary for TDP-43 aggregation. This conclusion does not exclude that possibility that aggregation might be initiated by covalently modified forms, with full-length forms subsequently aggregating by another process triggered by truncated aggregates. Such processes might include impairment of quality control mechanisms that establish and maintain proper TDP-43 conformation or that clear aggregated TDP-43. In this regard, one study found that truncated forms of TDP-43 expressed in cultured cells recruit full length TDP-43 into aggregates From the observation that apparently full length TDP-43 is present in aggregates, one can conclude that truncation cannot be essential for aggregation because the processes leading to truncation are presumably irreversible. By similar reasoning, the presence TDP-43 appearing to lack phosphorylation suggests that phosphorylation is not essential for pathological aggregation. However, unlike truncation, phosphorylation is not necessarily irreversible. It is conceivable that endogenous phosphatase activity might generate 43-kDa TDP-43 from the 45-kDa species (or other phosphorylated full-length species) after aggregation had occurred. We found no significant endogenous phosphatase activity upon TDP-43 in our post-mortem tissue samples, but this does not exclude the possibility that such a process was active Another possibility is that covalent modifications are not necessary to initiate and perpetuate TDP-43 aggregation. In the neurodegenerative conditions known as prion disease, the prion protein (PrP) converts to an aggregated form without covalent modification. Instead, pathological PrP aggregates seem to serve as a template for a conformational change in native PrP that drives further aggregation. Evidence exists for prion-like propagation of the aggregated proteins associated with several neurodegenerative diseases The significance of apparently aggregated 43-kDa TDP-43 in one of our controls in an amount approaching that found in in FTLD cases is uncertain. A separate preparation from the same brain gave similar results (data not shown). Histopathologic examination of this brain did not reveal TDP-43 positive intracellular inclusions, but biochemical techniques may be more sensitive that immunohistochemistry for the detection of pathological protein aggregates A waiver of approval was obtained from the Colorado Multiple Institutional Review Board because only autopsy tissue was used in these studies. Frozen blocks of human cerebral cortex from autopsy specimens were obtained from either the New York Brain Bank or the Neuropathology Division at the University of Colorado Denver. The clinical characteristics of all cases are summarized in Brain tissue was homogenized in 9 volumes ice-cold 25 mM Tris, pH 7.4, with Complete inhibitor cocktail (Roche). Where indicated, a phosphatase inhibitor cocktail, PhosStop (Roche) was also added to the homogenization buffer. Tissue was suspended in buffer using a 5-mL Tenbroeck tissue grinder on ice, then clarified by centrifugation at 600 g for 30 seconds. Total protein concentration of homogenates was determined by the bicinchoninic acid assay .This method is a modification of a technique used to demonstrate aggregated forms of yeast prion proteins We developed methods to isolate sufficient TDP-43 for further analysis from the high molecular weight regions of SDD-AGE gels . A largeThe gel was next modified to permit separation of a large volume of homogenate by vertical electrophoresis. First, the comb was removed. To accomplish this without tearing the delicate gel, the clamps holding the glass plates and spacers were loosened and removed. The gel assembly was held parallel to the floor, and the top glass plate was carefully slid away from the comb, without introducing air between the plate and the underlying gel, to expose the comb. The comb was then removed. Large volume wells were made by removing the agarose barrier between 4 adjoining wells with a clean micropipette tip. The top plate was then carefully slid back into position and the clamps were replace. In order to prevent the gel from sliding out of the bottom of the assembly during vertical electrophoresis, a 1 cm wide retention band was poured at the bottom of the gel: First, the assembly was held in an upright position until the gel gradually slid out from between the glass plates, exposing a 1 cm wide band. This was excised with a clean razor blade. The assembly was then inverted until the gel returned to its original position. The assembly was then placed horizontally on the bench top. Three strips of blotting paper, 1 cm ×14 cm were then insert into the 1 cm gap at the bottom of the gel. A 30% polyacrylamide solution (30% acrylamide (w/v), 0.6% bis-acrylamide (w/v), 20 mM Tris (pH 7.4) and 200 mM glycine, 0.1% SDS (w/v), 5 µL/mL 10% ammonium persulfate, 0.5 µL/mL TEMED) was then carefully pipetted onto the paper and allowed to fill the 1 cm gap by capillary action.When the polyacrylamide retention band polymerized, the gel was placed in the apparatus according to the manufacturers instructions. The buffer chambers were filled with a standard Laemmli running buffer. Samples were prepared by mixing 400 μg  of homogenate, typically about 100 μL, with an equal volume of 2× SDD-AGE loading buffer and incubated at room temperature for 10 min with 3 brief agitations using a vortex mixer. The electrophoresis was performed at constant 125 V with ice water cooling until a pre-stained 15-kDA marker  reached the top of the polyacrylamide strip. Regions of interest were excised from the gel with a clean razor blade. Up to 4 samples can be processed in parallel with this method using a single 2-gel apparatus. At least 1 control and 1 FTLD-TPD case were included in each run of the method.We modified the “three-layer sandwich gel electrophoresis” method Meanwhile, excised portions of large format SDD-AGE gels were weighed. SDS was added to 1% and dithiothreitol to 25 mM. Samples were boiled for 5 minutes to melt the agarose and to denature protein complexes contained within it. Ultrapure water was added to a final volume of 4 mL, and the samples were briefly boiled again. The hot agarose solution was then pipetted into the glass tube over the sealing layer of polyacrylamide. After gelling, the agarose was capped with 100 µL of a melted solution of 0.01% (w/v) bromphenol blue in 1% agarose (w/v).The tubes were loaded onto the Electro-eluter apparatus according to the manufacturers instructions, with the polyacrylamide sealing layer down. Electrophoresis was performed in Laemmli buffer at 15 V for 30 min, reduced to 5 V for 16 hr., then increased stepwise to 140 V over 5 hours. Under these conditions, bromphenol blue migrated to form a thin band just above the polyacrylamide sealing layer. The agarose column was extruded from the tube by running a 30-gauge needle of a 1 mL tuberculin syringe around the perimeter of the polyacrylamide plug to loosen its adherence to the glass tube, then pushing on the polyacrylamide plug with the thumb rest of the syringe. An approximately 1.5 mm wide slice of agarose encompassing the bromphenol blue band, and where appropriate, sequentially higher slices were excised with a clean razor blade for further analysis.SDS-PAGE was performed using standard Laemmli technique with precast tris-glycine gels (Invitrogen) For immunoblotting of both SDS-PAGE and SDD-AGE gels, proteins were transferred to polyvinylidene fluoride (PVDF) membrane using a mini-tank  using Towbin buffer per the manufacturers instructions For analysis of phospho-TDP-43 bands, blots were first probed and imaged using polyclonal antiserum 10782-2-AP as above. The blot was then stripped of antibody using a commercial stripping buffer  according to the manufacturers instructions. The blot was then probed with anti-phospho-TDP-43 (Ser409/Ser410), clone 1D3  and anti-rat IgG-HRP conjugate secondary antibody.SDS-PAGE gels for silver staining were run with unstained protein mass standards . Silver staining was done using a SilverXpress kit (Invitrogen) per the manufacturers instructions. Coomassie staining was done using Brilliant Blue G  following the protocol supplied by the manufacturer.Two brain homogenates were prepared from each of 3 FTLD cases. Tissue was homogenized using a 1 mL Tenbroeck tissue grinder on ice, in 9 volumes ice-cold 25 mM Tris, pH 7.4 with either Complete protease inhibitor cocktail or both Complete protease inhibitor and PhosStop phosphatase inhibitor cocktail. Samples were stored on ice while protein concentrations were determined, then 800 µg  aliquots of both protease inhibitor only or protease inhibitor and phosphatase inhibitor treated homogenates either stored on ice or incubated at 37°C for 1 hour. The samples were then subject to large volume SDD-AGE followed by concentration of the aggregate region, then separated by SDS-PAGE as described above. Immunoblots were made using polyclonal anti-TDP-43 antiserum. The 43-kDa and 45-kDa bands were quantified as described below, and ratio of the 43-kDa band to the 45-kDa band was determined.Urea was prepared in 0 M, 2 M, 6 M and 8 M concentrations in 50 mM Tris pH 6.8, and 15 µL of the urea solution was mixed with 5 µL of a 10% brain homogenate, prepared as described above, from an FTLD case. The mixtures were vortexed briefly and incubated for 60 minutes at room temperature. The samples were then processed for SDD-AGE and immunoblotting. To assess the effect of boiling in the presence of SDS and DTT, 5 µL of 10% brain homogenate was mixed with 5 µL of SDD-AGE loading buffer. One sample was held at room temperature for 10 minutes (per SDD-AGE protocol described above) while the other was heated to 100°C for 5 minutes. The samples were then processed for SDD-AGE and immunoblotting.All immunoblots were either directly imaged in a digital format, using a Kodak Image Station 4000  cooled-CCD digital camera, or converted to digital images from photographic film  by taking digital photographs of film images on a trans-illuminating stage . Digital images of Coomassie and silver stained gels were obtained by photographing them on a trans-illuminating stage.For quantitative analysis, digital images were imported into Mathematica software st (linear) or 3rd order polynomial curves were fit to the standard density dilution plots using the Mathematica “FindFit” function. The order of the fitted curve was chosen empirically to minimize residuals. In most cases photographic film showed a sigmoidal response best fit to a 3rd order curve, while the directly obtained CCD camera images showed a linear response. The equations for these fitted curves were applied to the apparent densities of the cognate bands in the experimental blot, so that quantities were determined relative to the dilution of the standard. This relative quantitation was used in comparisons between samples in different experiments. Absolute TDP-43 quantities were not determined. In measuring densities of bands, median pixel values were used instead of mean values because this method was less sensitive to irregularities such as specks and blank spots in the bands images. Particularly when measuring closely apposed bands, such as the 43-kDa and 45-kDa species, the measured density depended on the precise positioning of the region of interest. To minimize error and variance due to positioning the region of interest, each ROI median density was measured 5 times and the mean value determined.Plots of apparent density versus dilution or quantity of the standard were prepared for each band of interest. Either 1To determine yields from the concentration process that we developed, portions of an SDD-AGE gel region concentrate were loaded onto a PAGE gel with serial dilutions of a standard. In the case of aggregate region concentrates, whole brain homogenate of known protein concentration was the standard. In the study of the efficiency of pre-stained marker recovery from the gel, the same lot of pre-stained markers was the standard. The pre-stained marker gel was imaged unstained, otherwise the PAGE gel was silver stained. A plot of standard dilutions versus mean whole lane density was made in Mathematica, and the concentration of the aggregate region concentrates determined by fitting to this curve, using the method described above for determining individual band densities. Yields were then calculated from these concentrations.Statistical tests were performed using Mathematica software. Comparison of band densities between FTLD cases and controls were made using the Mathematica “TTest” function, which performs a Student T-test on univariate data such as in this study. Because it was not clear that the density values would be distributed normally, a non-parametric test was also applied. In all cases the T-test yielded the highest, i.e. least significant therefore most conservative, P value and this was reported.


Dinoderus minutus (bamboo borer) is a wood boring beetle that seriously damage dried bamboo and finished bamboo products. Management of D. minutus using pesticides showed negative effects on environment despite being very costly. By understanding influence of natural climatic conditions on their reproductive behaviour, could help us to develop a cost effective and environmental friendly strategy to cope up with this problem. In the present study, reproductive parameters and egg development of the bamboo borer were determined at 20%, 40%, 56%, 75% and 85% r.h. levels at constant temperature of 30° ± 2°C with 8 L-16D photoregime. From the results, eclosion to first instar larva was recorded at all relative humidities tested. The lowest shortest percentage of hatchability was recorded at 20% and 85% relative humidity with a mean incubation period of 4.63 ± 0.25 and 10.43 ± 0.32 days, respectively. It was noted that pre-ovipositional period decreased from 14.20 ± 0.49 to 7.20 ± 0.31 days as relative humidity increased from 20% to 75% and slightly increased to 8.00 ± 0.37 days at 85% relative humidity. We conclude that female beetles may have a particular hygropreference in oviposition as total egg production increased with increasing relative humidity.Wood products are highly exposed to infestation by powder post beetles.  Plants are always under the influence of biotic as well as abiotic stressful conditions  and Oryzaephilus surinamensis (Coleoptera: Cucujidae) showed increased insect mortality as relative humidity decreased , which consists of anhydrous molecules, and with the amount of cuticle, which has a lower water content than other tissues. Environmental temperature and humidity affects the developmental phase and transpiration through insect body surface , insects or their eggs may drown or be infected more readily by pathogens . This study would help us to develop cost effective and environmental friendly strategies to control insect pests without any health hazards.The present study was concentrated to understand the effect of relative humidity on egg hatchability and reproductive capacity of bamboo borer D. minutus were collected from infested bamboo culms in Bamboo Incubation Center and Handicraft in Simpang Pertang, Negeri Sembilan, Malaysia (2°57’25.61”N 102°18’12.69”E). Most bamboo culms infested by D. minutus belongs to the species Gigantochloa scortechinii, which is an important species of bamboo in Malaysia. Infested bamboo culms were kept in a plastic container with dimension of 30 cm × 20 cm × 25 cm kept in insect rearing room at 75% r.h. and 25 ± 3°C with 8 L-16D photoregime. Newly emerged beetles were collected and introduced into a new culture jar with cassava block using standards techniques of culture described by Abood et al. , Sodium iodide (NaI), Sodium bromide (NaBr), Sodium chloride (NaCl) and Potassium chloride (KCl). The relative humidity was obtained by filling solutions at the bottom of desiccators and monitored using a digital thermohygrometer. Throughout the study, investigation on reproductive parameters and egg hatchability were carried out in Petri dishes kept in desiccators corresponding to each relative humidity and hermetically closed. The desiccators were put into an incubator with controlled temperature of 30 ± 2°C.Different salt solutions and distilled water were used to control the relative humidity to which eggs and adult beetles were exposed. As indicated by Winston and Bates , Roca anNewly emerged beetles less than 7 days old were used to determine the effect of different relative humidities  on reproductive parameters at constant temperature of 30° ± 2°C with 8 L-16D photoregime. Ten pairs of adult beetles were exposed at each relative humidity tested. Each pair of adults were selected and separately introduced into Petri dishes lined with cassava pellet and filter paper. Observation on egg production was then started after 24 hours of setup. Cassava pellets were removed and broken up daily to assess number of eggs laid. The cassava pellet was replaced each time after inspection on egg production. Data collection on adult longevity and fecundity were collected until complete mortality.D. minutus were obtained from an established D. minutus culture on cassava. A total of 50 eggs were placed equidistant into Petri dishes lined with black filter paper for each relative humidity tested. Each Petri dish was kept in desiccators corresponding to each relative humidity and hermetically closed. The desiccators were put into an incubator with controlled temperature of 30 ± 2°C. Petri dishes were kept under these conditions and observation on egg eclosion and incubation period were observed daily under stereomicroscope and compared. During observation, eggs were removed as required with respect to age in days with a fine brush. Each egg was mounted on a concave microscope slide with a cover slip. This method was carried out to minimize the effect of relative humidity during the non-test conditions.The effect of relative humidity on egg hatchability and incubation period was inspected under different relative humidity levels  at constant temperature of 30° ± 2°C with 8 L-16D photoregime. Newly laid eggs of Statistical analysis software (SAS version 9.2) was used for the analysis on egg hatching, reproductive capacity and adult longevity at different relative humidities. One Way Analysis of Variance (ANOVA) and Fisher’s least significant difference (LSD) were used to test the effect of relative humidity on reproductive parameter and incubation period.D. minutus. There is no significant difference between incubation period at 40%, 56% and 75% relative humidity . The lowest and highest relative humidity caused a significant decrease in egg hatchability when compared with the intermediate relative humidity levels .The results presented in Table Highest eclosion to first instar larva was recorded at 75% relative humidity with 86% eclosion to success and a mean incubation period of 5.44 ± 0.14 days. Lowest relative humidity of 20% showed only 8% eclosion to success with a mean incubation period of 4.63 ± 0.25. Percentage of successful eclosion and incubation period increased with increasing relative humidity to 75%. However for 85% relative humidity, the percentage of successful eclosion decreased to 46%. This shows that egg development was affected by extremes in humidity (Plate 1). Longest incubation period was recorded at 85% relative humidity with a mean of 10.43 ± 0.32 days.D. minutus reproductive capacity  and adult lifespan . However, there is no significant difference in pre-ovipositional period between 56% and 75% relative humidity levels . Female D. minutus showed shorter pre-ovipositional period with increasing relative humidity until 75%. Ovipositional period  and mean number of eggs laid  showed no significant difference between 75% and 85% relative humidity levels. Shortest ovipositional period was recorded at 20% relative humidity with a mean of 3.8 ± 0.64 days while the longest ovipositional period was recorded at 85% relative humidity with a mean of 56.50 ± 1.21. Mean number of eggs laid increased from 3.20 ± 0.31 to 56.50 ± 1.21 from 20% to 85% relative humidity, thus indicating a preference for higher relative humidity conditions. Similar increment was recorded on mean ovipositional period which showed an increase from 3.8 ± 0.64 to 54.80 ± 0.94 days. The pre-ovipositional period decreased with increasing relative humidity levels from 20% to 75%. However, between 75% to 85% relative humidity, there was a significant increase in pre-ovipositional period. Adult lifespan for both sexes increase with increasing relative humidity. Female D. minutus had longer lifespan compared to male beetles under all relative humidities tested. There was no significant difference between adult lifespan for male  and female  between 85% and 75% and 75% and 56% relative humidity, respectively. Earliest mortality for adult beetle was recorded at 20% relative humidity for male and for female beetle on day 16 and 18, respectively. Longest lifespan for male beetle was recorded at 67 day at 75% relative humidity while longest lifespan for female beetle was 74 days at 85% relative humidity.Boring activity on cassava pellet was recorded within 24 hours of exposure under each relative humidity. Results in Table D. minutus under different relative humidity are shown in Figure Fecundity curves for Dinoderus minutus Fabricus).In order to establish a sustainable pest management, it is essential to know the physiology as well as reproductive capacity of pests viz-a-viz their relations with various natural climatic conditions. Thus present study was undertaken to investigate the effect of humidity on egg hatchability and reproductive biology of the bamboo borer  .From the observations in this study, most eclosion to first instar larva of D. minutus showed heterogeneous ovipositional pattern with most eggs being laid at higher relative humidities and the mean number of eggs laid increased from 3.20 ± 0.31 to 56.50 ± 1.21 at 20% to 85% relative humidities, respectively. The effect of relative humidity on oviposition indicates that females may have a particular hygropreference in oviposition as total egg produced and ovipositional period increased with increasing relative humidity. However, observation on egg hatchability showed that high relative humidity of 85% resulted with low egg hatchability (Table D. minutus was capable to reproduce at high relative humidity at early infestation in bamboo storage. As relative humidity decreased during the processing of bamboo culm, egg hatchability increases causing a pest outbreak. Romoser and Stoffolano (Xylocoris flavipes decreased at high relative humidity of 90%. David and Ananthakrishnan (Locusta spp. Andrewartha and Birch (D. minutus response towards relative humidity was found to be similar to the rice weevil, Sitophylus oryzae, an important pest in stored product. Fecundity of S. oryzae increased with increasing moisture content of wheat from 34% to 70% relative humidity (David and Ananthakrishnan, Female offolano  stated toffolano  showed tkrishnan  suggestend Birch  reportedD. minutus egg hatchability showed high mortality at 20% and 85% relative humidity levels compared to that between 40% to 75%. Lowest egg hatchability was recorded at 20% and 85% relative humidity levels. D. minutus egg suffers from loss of moisture that leads to shrinkage of both the embryo and chorion which prevents larval release. High relative humidity at 85% resulted in egg mortality due to excessive moisture. Reproductive capacity and incubation period of D. minutus increased with increasing relative humidity. Mean number of eggs laid increased from 3.20 ± 0.31 to 56.50 ± 1.21 from 20% to 85% relative humidity. Female beetle preferred to lay eggs in higher relative humidity as the number of eggs increased with increasing relative humidity. Pre-ovipositional decreased with increasing relative humidity. However at 85% relative humidity, pre-ovipositional period increased slightly compared to 75% relative humidity. Ovipositional period prolonged as relative humidity increased. Adult female showed higher fecundity characteristics with increasing relative humidity. Mean ovipositional period increased from 3.8 ± 0.64 to 54.80 ± 0.94 days with increasing relative humidity. The present study would help scientist to work out the strategy/s about the biological control of pests without causing any damage to health, environment as well as economy.The effect of relative humidity on