Here, we report the development of a new polyacrylamide (PAAm)/starch nanofibers’ blend system and highlight its potential as substrate for efficient enzyme immobilization. PAAm was synthesized and blended with starch. The final blend was then electrospun into nanofibers. The response surface methodology was used to analyze the parameters that control nanofiber’s diameter. Electrospun mat was then modified either by cross-linking or phytase immobilization using silane coupling agent and glutaraldehyde chemistry. Physico-chemical properties of blends were investigated using spectroscopic and thermal studies. The evaluation of immobilized enzyme kinetics on both pure and the starch blended PAAm nanofibers was performed using Michaelis–Menten kinetic curves. Fourier transform infrared spectroscopy results along with differential scanning and X-ray diffraction confirmed that blending was successfully accomplished. TGA analysis also demonstrated that the presence of starch enhances the thermal degradability of PAAm nanofibers. Finally, it was shown that addition of starch to PAAm increases the efficacies of enzyme loading and, therefore, significantly enhances the activity as well as kinetics of the immobilized enzyme on electrospun blend mats. There has been a great progress in the preparation, function, and evaluation of biomaterials concerning multifaceted practicability of them. Thanks to the outstanding properties and the key role in diversified areas of performance, a wide range of biomaterials has been increasingly used in biological applications. The acknowledgement that investigation of the impact of natural biopolymer-biomaterial along with its relevant mechanical and biological experiments comprising enzymatic survey can be amply efficient stimulates the authors to pursue this novel subject.Chemical and physical properties of starch have been widely studied due to its flexibility for a variety of applications because of its biodegradability and economical availability  which is one of the most important superabsorbent polymers. PAAm has a very routine synthesis protocol and it is known as one of the economical biocompatible polymers. PAAm was selected to be compounded with starch. It is supposed that its ability to absorb a large amount of water to form a stable hydrogel makes PAAm a promising candidate for developing biological applications and immobilization of aqueous enzyme’s solutions  method to investigate the effect of addition of starch (interconnected factor) on the diameter of PAAm nanofibers (selected response). DOE fits experimental results to mathematical equations representing some models based on a combination of values to predict the process (Chow and Yap Escherichia coli DH5α and primers for PCR were purchased from Sinaclone Co. The sequence encoding Aspergillus niger Phytase (P34752) was synthesized by ShineGene Molecular Biotech . Other analytical grade chemicals were obtained from Merck Co.Acrylamide monomer (AM),  was used for polyacrylamide (PAAm) synthesis. Potassium peroxodisulfate (KPS),  was used as a radical initiator for the synthesis reaction of PAAm. 3-Aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were bought from Sigma-Aldrich Co and PanreacQuimica San Co. for cross-linking reaction of nanofibers, respectively. Wheat starch was a general food grade prepared from the market. Formic acid was purchased from Riedel–de Haen as a solvent for electrospinning. Sodium phytate (P3168) was bought from Sigma-Aldrich Co. as a substrate of the phytase. The experiments were conducted to determine the role of adding starch in controlling the morphological properties of nanofibers. The effects of polymer weight fraction and blended starch percentage as independent variables and functional relationship between them attributed to molecular weight, viscosity, conductivity, and surface tension on the morphology of nanofibers were studied using RSM. Experimental designs were carried out by choosing the range of the weight fraction of polymer from 1.5 to 4 wt% and an appropriate proportion of blended starch from 20 to 50%. The central composite design (CCD) was used as an experimental design to obtain equal prediction in all directions from the center in which the variables vary in 5 levels with 5 axial points and 5 replicates at the center. Totally, it was indicated that 13 experiments were required to fit a second-order polynomial model , was added to the solution and free radical polymerization was started after initiator was added to the monomers. After 2 h, the polymerization was stopped and the resultant product was precipitated with acetone and preserved in vacuum desiccators.2 grounded static vertical collector covered by a piece of aluminum foil was used for the fiber deposition. The complete electrospinning apparatus was enclosed under a vacuum hood and an electrospinning process was carried out at room temperature.We made starch/PAAm solutions according to the experimental design Table  by dissoAspergillus niger Phytase was transformed to the competent cell of E. coli. After colony PCR screening, plasmids were purified from the positive clones by the alkaline lysis method and then digested with HindIII/BamhI. The phytase gene was inserted into pFPMTMFα as an expression and also as a shuttle vector. pFPMTMFα comprising phytase was transformed into E. coli DH5α. All the cloning steps were done according to Sambrook et al.  at 37 °C for an overnight (16–18 h) on a rotary shaker at 250 rpm. The main culture medium  was inoculated with 5–10% (v/v) from pre-culture after washing by sterile water. The main cultivation for enzyme production was performed for 72 h under the same conditions as with the pre-culture. The cells were harvested by centrifugation at 2500×We test the phytase assessment by measuring the amount of inorganic phosphate liberated from sodium phytate  of immobilized phytase mixtures was measured via a UV–Vis Spectrometer  at the absorbance of 380 nm. Michaelis–Menten kinetics for immobilized and free phytase were measured for different phytic acid concentrations as substrate.The morphology of the electrospun blended PAAm/starch nanofibers was characterized with a high-resolution scanning electron microscopy (SEM) (Hitachi S4160); and ImageJ software was used for measurement of the fibers’ diameter. Design Expert software  was used for evaluation of the fibers’ diameter based on the affected nanofibers variables. Fourier transform infrared spectroscopy  was used to study differentiations of spectra for starch, PAAm, and blended PAAm/starch which could indicate the changes in the chemical structure upon blending. Thermal analysis of the fibers was done using differential scanning calorimetry (DSC) (NETZSCH 200 F3) and thermal gravimetric analysis (TGA) (Linseis PT-10). DSC measurement allows one to evaluate the thermal behavior differences of the nanofibers and determining the influence of starch on PAAm nanofibers, before and after cross-linking modification. About 11 mg of nanofibers were heated up at 10 °C/min from room temperature to 400 and 800 °C at 10 °C/min under nitrogen atmosphere for DSC and TGA analysis, respectively. Wide angle X-ray diffraction  measurements were performed to obtain crystalline structure information. The X-ray generator was equipped with an iron tube operating at 35 kV and 25 mA. The wavelength of irradiation was ~0.194 nm. XRD diffractograms were acquired at room temperature over a 2SEM images show the random topography of the nanofibers. Influential parameters of the polymer molecular weight, polymer concentration, and polymer solubility in the solvent associate with dependent solution viscosity, conductivity, and surface tension which all directly affect the morphology of electrospun nanofibers  takes place at 1665.92 (cm−1) in the blended nanofibers  is attributed to the vibration of N–H bending of PAAm in the nanofibrous format, which is presented at 2961.55 (cm−1) in the blended nanofibers.FT-IR spectroscopy is used to investigate the spectra of the wheat starch, PAAm, and blended PAAm/starch nanofibers, as shown in Fig. −1) is attributed to the characteristic absorption peak of the stretching vibration of O–H. The peaks at ~1084.07 (cm−1), 1158.40 (cm−1) and 1019.60 (cm−1) are related to the vibration of C–O in C–O–H groups. The peak at 2928.39 (cm−1) is ascribed to the asymmetric stretching of C–H whose intensity vibration is significantly lessened in the blend format. The peak at 1646.55 cm−1 is attributed to the adsorbed water. The peak at about 1007.87 (cm−1) is ascribed to C–O–H stretching. The peaks at 857 (cm−1) and 763.81 (cm−1) are related to the C–H absorbance of d-glucopyranosyl ring stretching.In starch spectra, a strong and broad peak at 3423.18 (cm−1) and 2961.55 (cm−1) imply the presence of PAAm, and peaks at 796.40 (cm−1), 1019.60 (cm−1), and 1170.67 (cm−1) are attributed to the starch in the blended nanofibers. FT-IR investigation demonstrates that blending was carried out in a satisfactory way due to the presence of all the starch and PAAm bonds in the blended nanofibers.Peaks at 1665.92 (cmTpeak), onset peak temperatures, 50% mass loss temperature, % mass change between 300 and 600 °C, and % residue of nanofibers weight loss. Observing the percentage of mass loss between 300 and 600 °C proves that the most part of degradation related to starch/PAAm decomposition is occurred in this range. The analyzed temperatures decreased with the addition of the starch loading in PAAm nanofibers, indicating a destruction in thermal stability because of the starch incorporation.We characterize nanofibrous mats by thermogravimetric analysis (TGA) which is used to predict their thermal stability from the temperature of 25 °C up to 800 °C at heating rate of 10 °C/min under nitrogen atmosphere. Figure Comparing mass loss temperatures of the PAAm/starch nanofibers and cross-linked ones can barely specify that the cross-linking modification significantly increases the nanofibers thermal stability. However, immobilization procedures and putting nanofibers in the aqueous enzyme solution for 18 h at 4 °C decrease the thermal stability (in comparison with cross-linked nanofibers). This is indorsed by comparing 50% mass loss temperatures (T50% mass loss) of the PAAm/starch nanofibers, cross-linked PAAm/starch nanofibers, and immobilized phytase on the PAAm/starch nanofibers, which are 348, 500, and 411 °C, respectively. Cross-linking improved thermal stability by about 43% measured at T50% mass loss and also it modified 18% mass residue (in comparison with blended nanofibers) which could be regarded as an evidence for proper cross-linking procedure. Nevertheless, holding nanofibers at 4 °C for 18 h while shaking in enzyme solution reduces thermal degradation of them .According to TGA results, it is proved that the addition of starch reduces the thermal stability, since it increases the degradation of PAAm nanofibers as reported previously  of the starch by DSC is difficult. Moreover, the multiple phase transitions during the heating process and the instability due to evaporation of water included in starch make the study of thermal behavior and crystallography of starch materials more difficult   of the starch caused by losing its moisture.In pure PAAm, despite the fact that the peak centered at 273 °C, no significant melting point is observed due to the poor crystalline structure of PAAm which is proved by XRD analysis thereafter. In blended nanofibers, the presence of two peaks is observed and the rather huge exothermic peak centered at 100 °C is related to the starch and the peak which is shifted at 280 °C belongs to the PAAm , blended with different amounts of wheat starch and fabricated randomly oriented nanofibers. The SEM images and RSM diameter analysis have shown that more percentage of starch addition—proportionally against the percentage of PAAm—could lead to the reduction of the resultant nanofibers’ diameter. The blend was successfully made as the FT-IR results had proved the perfect structure. The thermal properties of PAAm nanofibers were shown to be improved by the presence of starch which enhanced the thermal degradability of PAAm nanofibers. Moreover, we have shown that cross-linking had synergetic effects in addition to surface modification which eventually enhanced thermal stability as well as preventing undesirable transformation; therefore, it increased the stability of nanofibers in unfriendly environment. This paper shows the promising potential of enzyme immobilization of starch/PAAm nanofibers. Overall, it was shown that the addition of starch could significantly increase the immobilized enzyme activity. We conclude that starch-aiding nanofibers improve kinetics activity of immobilized enzyme and also they surpassingly operate as a promising substrate for enzyme immobilization.


Self-regulated learning (SRL) skills are essential for learning during school years, particularly in complex problem-solving domains, such as biology and math. Although a lot of studies have focused on the cognitive resources that are needed for learning to solve problems in a self-regulated way, affective and motivational resources have received much less research attention. The current study investigated the relation between affect , motivation , mental effort, SRL skills, and problem-solving performance when learning to solve biology problems in a self-regulated online learning environment. In the learning phase, secondary education students studied video-modeling examples of how to solve hereditary problems, solved hereditary problems which they chose themselves from a set of problems with different complexity levels . In the posttest, students solved hereditary problems, self-assessed their performance, and chose a next problem from the set of problems but did not solve these problems. The results from this study showed that negative affect, inaccurate self-assessments during the posttest, and higher perceptions of mental effort during the posttest were negatively associated with problem-solving performance after learning in a self-regulated way. Problem-solving is an important cognitive process, be it in everyday life, at work or at school. Problem-solving is the process in which people put effort into closing the gap between an initial or current state  and the goal state . ResearcSelf-regulated learning skills are especially important in learner-controlled, online learning environments in which students need to be able to accurately keep track of their own learning process  and have to make complex decisions about what problem-solving task to choose next during their learning process . Apart from the high cognitive demands imposed by SRL, which have been investigated frequently in previous research e.g., , learninThere are many different kinds of problem-solving tasks, varying from well-structured transformation problems that have a clearly defined goal and solution procedure, to ill-structured problems that do not have a well-defined goal or solution procedure . In educFor learning to solve such complex problems, it is efficient to “borrow” and “reorganize” knowledge of others  by learnAccording to the resource-allocation framework by Despite this expectation, SRL skills, such as monitoring one’s own learning processes, have been found to be suboptimal when studying worked examples ,b, 2016.In a previous study by Students’ affect and motivation can facilitate or hinder students when learning to solve problems in a self-regulated way. Affect was found to influence the use of different strategies , SRL activities , and motivation; all factors that can impact academic achievement . MoreoveAccording to theories on SRL both affect and motivation play an important role in SRL e.g., . AccordiAlthough the study by In the domains of problem-solving and decision-making, it was found that positive affect facilitates flexible and creative thinking, and decision-making in complex environments such as medical decision-making . In a reIn contrast to the findings showing that positive affect can facilitate problem-solving performance e.g., , some stTo sum up, positive affect could facilitate problem-solving and decision-making. Yet, this seems to be dependent on the type of problems used in the different studies. The problem-solving tasks in the review by Self-determination theory SDT; ,b predicAutonomous motivation types are associated with better learning outcomes, persistence, and psychological well-being relative to controlled motivation types. Autonomous motivation types were found to be related to better text comprehension e.g.,  and selfIn summary, next to enhancing learning and problem-solving performance, autonomous motivation could also facilitate the use of SRL skills during learning. Furthermore, in multiple studies by The relation between affect, self-assessment accuracy, making complex decisions about the learning process , perceived mental effect and motivation was investigated in a learner-controlled, online environment, in which students could monitor and regulate their own learning. In this environment students first received video-modeling examples teaching them how to solve stepwise, hereditary problem-solving tasks, how to make a self-assessment , and how to select the next task . In each video-modeling example, after solving the problem, the model rated the perceived amount of invested mental effort , made a Figure 1). Task complexity of the well-structured problems was defined in terms of element interactivity: the higher the number of interacting information elements that a learner has to relate and keep active in WM when performing a task, the higher the complexity of that task and the higher the cognitive load it imposes  from the second year in the higher education track. All students gave their consent to participate in this study. Students’ parents received a letter in which information about the study was provided and parents were asked for their consent.Participants were 136 secondary school students  on a 5-point scale . For botThe pretest and posttest consisted of three well-structured problem-solving tasks about hereditary problems based on the laws of Mendel which differed in complexity in terms of element interactivity cf. . All proTwo video-modeling examples showed how to solve a hereditary problem step by step. The hereditary problems explained in the videos had a similar solution procedure because in both videos the goal was to find the genotype of the child based on information about the parents . The surface features were different between the problems explained in the videos . In the videos, a model was thinking aloud about how to solve the problem and wrote down the solution step by step. One video had a female model and the other video had a male model explaining how to solve a problem see , made a How much effort did you invest in solving this problem?’ Students could respond on a 9-point scale, ranging from 1  to 9 .After each posttest question, mental effort invested in solving the posttest problems was measured by asking: ‘n = 130).Students made a self-assessment of their performance as a measure of self-monitoring after each posttest problem-solving task cf. . StudentFigure 1, cf. Figure 1). The level of support was not included in the level of complexity. Note, during the posttest students did not actually study the tasks they choose and they were made aware of that. The mean regulation choice complexity score for the posttest was calculated. There were 33 students who did not make a regulation choice and therefore they were left out of the analysis of regulation choice data (n = 103).During the posttest, the complexity of the regulation choices of students was measured. Students could choose problem-solving tasks to study next from a database with 75 problem-solving tasks at five complexity levels (see because I am supposed to do so”), (2) introjected , (3) identified , and (4) intrinsic motivation . Items were measured on a 5-point Likert-type scale ranging from 1  to 5 . The four subscales were combined into an autonomous motivation composite (intrinsic and identified motivation) and a controlled motivation composite  Cronbach’s alpha was α = 0.89. For the controlled motivation composite (n = 126) Cronbach’s alpha was α = 0.65.At the end of the study, all students filled out a 16-item task-specific version of the academic self-regulation scale . In four2. In Figure 2, the procedure of the study is depicted. First, all students filled out the affect questionnaire. Then they took the pretest which was followed by two video-modeling examples. Then students entered the SRL phase in which they practiced with four problem-solving tasks of their choice from a database with 75 problem-solving tasks at five complexity levels . Students also practiced with rating their perceived mental effort, self-assessment, and regulation choices. Then after practicing four problem-solving tasks, students took a posttest with three problem-solving tasks of different complexity. Students’ perceived mental effort, self-assessments, regulation choices, and problem-solving performance were measured. Finally, all students filled out the motivation questionnaire.In 50-min sessions in the computer room at their schools, students participated in the current study using an online learning environmentTable 1, the descriptive statistics of the pretest, posttest, perceived mental effort, self-assessments during the posttest , positive and negative affect scale, and autonomous and controlled motivation can be found. In Table 2, the correlations between these variables are shown. Pretest performance was significantly positively related to posttest performance. Positive affect was significantly positively associated with negative affect, indicating that students who scored higher on positive feelings also scored higher on negative feelings. Positive affect was significantly positively related to autonomous motivation. In line with Hypothesis 1b, negative affect was significantly negatively related to performance on the pretest and posttest, which indicated that students who reported more negative feelings scored lower on the tests.In In line with Hypothesis 2a, both self-assessment bias and absolute accuracy of self-assessments during the posttest were significantly negatively related to posttest performance. That is, the larger the difference between self-assessment and actual performance was, the lower posttest performance was. It seemed that students who are less accurate in their self-assessment also score lower on the posttest.In support of Hypotheses 3b and c, the ratings of perceived mental effort showed a significant negative relation with the self-assessment raw score, bias, and complexity of regulation choices. This means that students who experienced a higher mental effort showed lower self-assessment and bias values, and choose less complex tasks to restudy. Both self-assessment raw scores and bias were positively correlated to the complexity of regulation choices. That is, the higher self-assessment raw scores and bias were, the more complex regulation choices were. In line with theories of SRL, this shows the sensitivity of regulation choices in relation to self-assessments  = 8.59, p = 0.014. Wilcoxon tests were used to follow up this finding. It appeared that regulation choice complexity differed significantly between moments 1 (Mean rank = 2.14) and 3 (Mean rank = 1.83), T = 0.30, r = 0.21 . Yet, no significant differences between selection moments 1 and 2 (Mean rank = 2.03) or 2 and 3 were found.The complexity level at which students selected a task for restudy and how they performed on the different complexity levels in the posttest were explored. Regulation choice complexity was not normally distributed. The mode of all three selection moments was regulation choice complexity 1. Therefore, a Friedman’s ANOVA was conducted for regulation choice complexity at all three selection moments during the posttest. The regulation choice complexity differed significantly over the three moments, χF = 56.13, p < 0.001, M = 2.45, SD = 1.68) was significantly higher compared to task 2 , p < 0.001, and compared to task 3 , p < 0.001. There was no significant difference between performance on tasks 2 and 3.Furthermore, as a check on the complexity of the problem-solving tasks in terms of element interactivity, a repeated measures ANOVA with complexity levels as a within-subjects variable was performed. It showed that problem-solving performance on the posttest differed significantly between the complexity levels of the problem-solving tasks, We performed stepwise regression with pretest performance in Model 1 and positive affect, negative affect, self-assessment accuracy during the posttest, regulation choice complexity, perceived mental effort, autonomous, and controlled motivation in Model 2. We assessed multicollinearity in accordance with the guidelines by Table 3, Model 1 with pretest performance as a predictor of posttest problem-solving performance was significant, F = 8.80, p = 0.004, R2 = 0.08. Pretest performance was a significant positive predictor of posttest problem-solving performance.As shown in F = 4.89, p < 0.001, R2 = 0.30. Model 2 explained more variance compared to Model 1, ΔR2 = 0.22, p = 0.001. Pretest performance was again a significant positive predictor or posttest problem-solving performance in Model 2. In line with Hypothesis 1b, negative affect was a significant negative predictor of posttest problem-solving performance. That is, the more negative affect students reported, the lower their posttest performance was. Also, in support of Hypothesis 2a, self-assessment accuracy was a significant negative predictor of posttest problem-solving performance. Self-assessment accuracy during the posttest was measured as absolute accuracy. The lower this measure is the more accurate self-assessments were. Thus, the negative relation with posttest performance means that the less accurate students’ self-assessments were, the lower posttest performance was. Furthermore, in line with Hypothesis 3, perceived mental effort was a significant negative predictor of posttest problem-solving performance. That is, the higher perceived mental effort during the posttest was, the lower posttest problem-solving performance was.In Model 2, positive affect, negative affect, posttest self-assessment accuracy, posttest regulation choice complexity, posttest perceived mental effort, autonomous and controlled motivation were added as predictors, The current study investigated the relation between affect , SRL skills , perceived mental effort, motivation , and performance when learning to solve problems in a complex learner-controlled, online learning environment with secondary education students. Students performed worse on the more complex problems during the posttest. Also, regulation choice complexity was lower after the most difficult problem-solving task when compared to the least complex problem-solving task at the posttest. Interestingly, the results showed that students’ negative affect, SRL skills, and perceived mental effort play a crucial role in learning to solve problems in a self-regulated way in a learner-controlled study environment.In contrast to Hypothesis 1a, positive affect was not a significant predictor of problem-solving performance in the current study using well-structured problem-solving tasks with high element interactivity. This result does not fit previous findings showing that positive affect improves cognitive processing e.g.,  and acadIn line with Hypothesis 2a and theories of SRL e.g., , self-asIn contrast to Hypothesis 2b, the regulation choice complexity was not related to problem-solving performance. This might be explained by the way we operationalized regulation choices. That is, students had to choose what task they wanted to work on next. According to the discrepancy-reduction framework of regulation e.g.,  studentsPerceived mental effort during the posttest was significantly related to problem-solving performance which was in line with Hypothesis 3. That is, the more mental effort students experienced during the posttest, the lower their posttest performance was. This finding is in line with CLT e.g.,  and the Contrary to our expectations (Hypotheses 4a and 4b), motivation was not a significant predictor of problem-solving performance. Based on earlier studies e.g.,  autonomoLimitations of the current study are the small number of secondary education students who could take part in the study. Future research could replicate the current study with more participants. This would also enable researchers to take into account different motivational profiles and their relation to positive and negative affect as predictors of problem-solving performance. Also, problem-solving performance was quite low and measured using a limited set of tasks during the posttest. It would be interesting to use more tasks for a longer period of time covering the SRL phase and posttest to investigate the effect of motivation and affect. We found positive and negative affect to be positively related which could have been caused by the intensity of affect . Future In conclusion, the current study showed that negative affect, monitoring accuracy, and perceived mental effort are predictors of problem-solving performance of secondary education students learning to solve problems in a learner-controlled, online environment. The fact that these predictors were all negatively related to performance is an important indication that students need more support when learning to solve problems in a self-regulated way. Interventions to support SRL processes (e.g., training, cf. In accordance with the guidelines of the ethical committee at the Department of Psychology, Education and Child studies, Erasmus University Rotterdam, the study was exempt from ethical approval procedures because the materials and procedures were not invasive.MB, LW, and FP worked together on the theoretical framework and the design of the study. Both MB and LW prepared the materials, and collected the data. MB, LW, and FP worked together on the analysis of the data. Writing the manuscript was done by MB, LW, and FP in collaboration. Finally, MB, LW, and FP approved the manuscript and are accountable for it.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Impact: what every scientist seeks in their career. It is a sign of widespread influence, a reflection of the field's continued interest, and validation of our ideas. The impact of a scientist's work is often expressed using simple numbers found in citation reports, journal impact factors, and h-indices, but perhaps the truest measure is the respect of their peers. By this metric, the impact of Dr. Marie T. Filbin's life and career was profound, as evidenced by the many articles published by friends and colleagues in remembrance of Marie since her passing  myelin inhibits axonal regeneration was first proposed in the early 1980s Berry, , and MarBaldwin and Giger. The next chapter is a historical and personal overview of the myelin-associated inhibitor field by McKerracher and Rosen. It is followed by a review from Soheila Karimi-Abdolrezaee's lab that describes the pathophysiologic events that affect oligodendrocytes after CNS injury, as well as cellular approaches currently used to promote remyelination . This topic is examined from a neuronal perspective by Kaplan et al. in their discussion of how neuron-intrinsic factors contribute to both axonal regeneration and inhibitory factor neutralization in the extracellular environment , which serves as a fitting counterpart to the review by Rao and Pearse that comprehensively describes how specific axon-glia signaling pathways influence both myelination and axonal regeneration.This e-book begins with several reviews that expand on the theme of myelin and its function in the injured CNS. While myelin-associated inhibitors are typically viewed in a negative light, these proteins do have normal physiologic roles that have been discussed by Carmel et al.). Several genes identified in this screen—arginase I, interleukin 6, secretory leukocyte protease inhibitor, and metallothionein I/II—were shown to overcome MAG inhibition in their own right, and their effects and mechanisms are discussed by Siddiq and Hannila.In 1999, the Filbin lab made another seminal contribution to the axonal regeneration field when they reported that elevation of cyclic AMP (cAMP) in postnatal rat neurons was sufficient to overcome inhibition by MAG and CNS myelin . While this story would appear to bring Marie's career full circle, it is in fact a reminder of all that remains unknown in this field and the work that still lies ahead. New discoveries regarding the effects of MAG, Nogo, and other myelin-associated inhibitors will undoubtedly provoke debate, and that is something Marie would have welcomed.Fittingly, we end with a contribution from Marie's laboratory on the subject that first brought her to prominence: MAG biochemistry and function. As a member of the Siglec family, it is well known that MAG can bind complex gangliosides such as GT1b and GD1a (Kelm et al., Frontiers in Molecular Neuroscience for their support and guidance throughout this process, one that has been personally and scientifically rewarding for both of us.She also would have been deeply grateful to all of the distinguished scientists who contributed to this Research Topic, and we extend our deepest thanks to each of them for their time and efforts. We also would like to thank the reviewers for their input and the staff of All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This method was applied to analyze the molecular junctions comprising various thiol molecules on a gold substrate, revealing that the noise in the junctions mainly came from the fluctuation of the thiol bonds. Furthermore, we quantitatively compared the frequencies of such bond fluctuations in different molecular wire junctions and identified molecular wires with lower electrical noise, which can provide critical information for designing low-noise molecular electronic devices. Our method provides valuable insights regarding noise phenomena in molecular wires and can be a powerful tool for the development of molecular electronic devices.We report a noise mapping strategy for the reliable identification and analysis of noise sources in molecular wire junctions. Here, different molecular wires were patterned on a gold substrate, and the current-noise map on the pattern was measured and analyzed, enabling the quantitative study of noise sources in the patterned molecular wires. The frequency spectra of the noise from the molecular wire junctions exhibited characteristic 1/ Molecular wires (MWs) are the basic building blocks of molecular electronic devices12345123456789101212131515f2 noise behavior regardless of MW species, unlike metal-metal nanojunctions showing common 1/f noise. This behavior was used to identify electrical signals originating from the MWs. Furthermore, the method was used to study the MW junctions of various thiol molecules on a gold substrate. The results indicate that the electrical noise in the junctions mainly originated from the fluctuations of thiol bonds between the MWs and the gold surface. In addition, we quantitatively compared the bond-fluctuation frequencies of different MWs and identified the MWs with lower noise. These results show that the electrical fluctuations measured by our method can provide useful insights regarding the physical properties of MW layers and clues to understand the behavior of individual MWs. Considering that the electrical noise has been one of the most difficult problems to study and is critical for molecular electronic devices, our results are expected to be a major breakthrough in molecular electronics research and may have a significant impact on the development of molecular electronic devices.Herein, we report a noise mapping strategy to reliably identify and map the sources of electrical noises in various MW junctions. In this strategy, we patterned different MW junctions in a single metal substrate via a microcontact printing method and mapped the electrical currents and noise power spectra of the sample. Here, the different MW junctions were based on the same substrate and mapped via a single scanning probe, minimizing the possible variations from the substrate and probe and enabling reliable studies on the effect by MWs in the junctions. Analysis of the mapping data indicated that the MW junctions exhibit a characteristic 1/current and PSD maps were analyzed to obtain maps of the resistances and the mean-square fluctuations of resistances of the patterned MWs, respectively. Note that the characteristics of the scanning probe-based MW junctions could vary due to the variations of the probes and substrates used. However, with our strategy, we could measure the noise characteristics of different MWs using a single probe on the same substrate. Thus, the possible variations of the MW junctions, which have often been a serious problem in previous works, were minimized, enabling reliable comparative studies of the different MWs.C9 and gold regions were ~4 and 8 μm, respectively. The measured height of the C9 monolayer was approximately 1.1 nm, which is consistent with the reported length of a C9 moleculeSI) at a specific frequency while measuring the topography and current maps via noise microscopy. We obtained a current-normalized noise PSD (SI/I2) map by dividing the SI values in the SI map by the corresponding current (I) values in the current map. The SI/I2 value is a useful parameter to represent the noise level of an electrical channelSI/I2 map at 31.6 Hz. The noise level of the MW (~10−5 Hz−1) was considerably higher than that in the bare gold region (~10−9 Hz−1). The low noise level in the gold region indicated negligible background noise in our noise microscopy system. In contrast, the high noise level in the C9 monolayer region implies that the C9 MWs between the gold and AFM probe were generating a significant amount of electrical noise. As a control experiment to check the reliability of our electronics, we used our electronics (without the AFM system) to measure the electrical noise from a dummy resistor (1.0 GΩ glass glaze film resistor) whose resistance was similar to that of the C9 MW junction , the PSD measured at the gold region showed a typical 1/f noise behavior with β ~1. In contrast, a 1/f2 noise behavior with β ~2 was observed when we measured noise spectra in the C9 region.In our setup, we placed a conducting probe at a fixed location on the MW-patterned sample and measured the noise spectra from that location. C) and non-conducting state (N), the electrical noise generated by the noise source should have a Lorentzian form like1520Previous works have shown that if a noise source can generate a two-state conductance fluctuation between a conducting state (τ is the characteristic time of the fluctuation and is defined as (λ + μ)−1, where λ and μ are the transition rates of the C-to-N and N-to-C transitions, respectively. At a rather high-frequency condition of 2πfτ ≫ 1, the PSD can be approximated to be proportional to 1/f213f2 noise in the C9 monolayers could be generated by the uniform Lorentzian noise sources in the MW layerτ as 11 ms  and gold region (β~1.0). We obtained consistent results from all different MW samples presented in this manuscript, indicating that the scaling analysis of the noise spectra is a simple yet powerful means to identify the noise generated by MWs. Considering that it is often difficult to distinguish the noise generated by MWs from that generated by metal electrodes in MW junctions, this result can provide an important tool for the research concerning charge transport phenomena in MW junctions.We measured the scaling factor ctor map . Here, t1625A and a known bias voltage (V) was used to measure the electrical currents (I) through a layer of MWs, the measured junction resistance (Rjunction) of the substrate-MWs-probe junction can be written using the resistance of a single MW (RMW) as follows :When a conducting probe with a contact area n represents the number of MWs in the unit area of the MW layer. Inversely, the RMW could be obtained from the Rjunction (=V/I) as follows:where n value has been reported as ~4.65 molecules/nm2 A of the conducting probe is still an unknown parameter. However, because we used the same probe to measure the electrical currents from different MWs  in the different molecular layers (RMW) as R for simplicity. The single-molecule resistance values of C8, C9 and C11 MWs were ~0.9, 4.5 and 22.5 GΩ, respectively. The scaling factor β of the noise-frequency spectra measured on these different MW regions exhibited a value of approximately 2.0, indicating that we were measuring the currents and resistances originating from the MWs rather than metal-metal nanocontacts. In our experiment, because a single probe was used to measure the resistance values of different MWs under the same conditions, the possible variation of the MW junctions was minimized, and we can precisely estimate the ratio of the molecular resistance values of the different MWs in Using the current map in r layers . In the τ. At a rather high frequency f, the PSD (SR) of such resistance fluctuations in the MW junction can be written as30The electrical noise generated in the MW junctions is an important parameter that should determine the performance of MW-based devices. In addition to the electrical currents, we also measured the map of the electrical current noise in the layers of different MWs . Further15<δR2> and f0 are the mean-square fluctuations of the molecular resistance and 1/(2πτ), respectively. Previous studies have shown that electrons tunneling through an MW junction can provide energy to the junction by interacting with phonons21B, f0 can be written as30Here, I denotes the current through an MW. Using f0 and I, we calculated the constant B as ~1.3 × 104 Hz/nA. At high-frequency conditions of f/f0 >>1, Here, The noise PSD values of the fluctuations of the resistance and current through an MW are related by20<δR2> can be written asand from <δR2> obtained after using SI/I2 map in SI and I values should be proportional to the number of MWs (nA) between the probe and goldSI/I2 values should be proportional to 1/(nA). Thus, we multiplied the values in the measured SI/I2 map by nA to obtain SI/I2 values for an ‘individual’ MW. In addition, the current through a single MW was obtained by dividing the current values in the measured current map by nA. The <δR2> map shows the clear differences between the <δR2> levels for different MWs. The <δR2 > values in the C8, C9 and C11 regions were ~7.6 × 1017, 6.7 × 1018 and 4.8 × 1020 Ω2, respectively. We are the first to have quantitatively measured and compared the mean-square fluctuations of the molecular resistances of different MWs, which should be a significant breakthrough for noise studies on MWs.<δR2> values as a function of the molecular resistance R. Here, the <δR2> value of the 4-mercaptopyridine (MPD) MW was estimated from the SI/I2 map measured on an MPD/C9 patterned sample  asd sample . As indid can be written as31Although the MPD and other alkanethiol MWs had completely different molecular chains with different lengths and structures, they still exhibited a similar behavior. Previous works have shown that the resistance of an insulating MW with length R0 corresponds to the contact resistance, which should depend on the metal-molecule bonds and the contact between the molecules and AFM probeR0 should be highly similar for all MWs in the graphα is the tunneling attenuation factor, which depends only on the chain of the MWs<δR2> of the molecular resistance values, which is responsible for the current noise in the MW junctions, can be written asHere, Here, the first term originates from the molecular contact resistance and its fluctuations32SI/I2) spectra that were measured in an MPD region (blue line) and a C9 region (red line). Both regions exhibited 1/f2 noise characteristics. The PSD spectra on the MPD and C9 regions were fitted well using Lorentzian curves (black dashed lines) with fitting parameters τ of ~1.3 ms and 11.5 ms, respectively. The results are compatible with previously reported values13τ is the characteristic time, which should be proportional to the mean time interval between each bond-fluctuation event. Thus, the shorter τ of the MPD molecules indicated that the bond-fluctuation events occurred more frequently in the MPD molecules than the C9 molecules. One plausible explanation is the larger currents in the MPD molecular regions because the energy needed for the bond fluctuations is largely provided by the current passing through the MW junctions21The spectral analysis of the noise spectrum was used to reveal the more detailed characteristics of the MW-based junctions . Figure τ on the molecular layers (Eq. (8)τ can be written asFor a more detailed analysis, we obtained the map of r layers . By inseτ on each point of the MPD/C9 patterns to obtain a τ map 21B represents the frequency of the molecular bond fluctuations when a specific amount of electrical current passes through, and thus, it can be a useful characteristic parameter for demonstrating the noise properties of specific MWs. We estimated the map of B on the MPD/C9 patterns using the τ map, the current map and B in the MPD and C9 regions were ~1.52 × 103 and 1.29 × 103 Hz/nA, respectively. These results indicate that with the same amount of current, the molecular bonds of the MPD molecules fluctuate more frequently than those of the C9 molecules. A previous study suggested that the characteristic frequency of the noise in molecular layers will depend on how densely the molecules cover the gold substrate21B value of the MPD molecule in our data could be attributed to the lower packing density of the MPD molecules than that of the C9 molecules. Our results illustrate that we can expect more molecular bond fluctuations and thus electrical noise with a higher frequency in the MPD-based electrical junctions than in the C9-based electrical junctions. The noise characteristics of the MWs directly affect the quality of the MW junctions and eventually determine the performance of the MW-based devices. Our method allowed us to quantitatively compare the characteristic parameters determining the electrical noise in MW junctions and even identify the MW species with lower noise under the same conditions. These results should provide an important guideline for developing low-noise molecular electronic devices.Here, the proportionality factor electrical currents and noise PSDs were obtained and used for the comparative analysis of the electrical resistance values and the noise characteristic parameters in different MWs, respectively. Importantly, we observed a characteristic 1/f2 noise behavior in the noise PSDs measured from the MW junction, whereas a simple metallic junction exhibited a typical 1/f noise behavior. This characteristic behavior was used to qualitatively distinguish the electrical signals of the MW junctions from those of metallic junctions. In addition, we successfully mapped the mean-square fluctuations <δR2> of the molecular resistances in thiol MW junctions and found that <δR2> was proportional to the squared molecular resistance R2, indicating that the noise in the junctions originated mainly from the fluctuation of the thiol bonds on the Au electrode surface. Furthermore, we quantitatively compared the frequency of such bond-fluctuation events and identified MWs with lower electrical noise, which could be an important guideline for designing low-noise molecular electronic devices. Considering that it has been extremely difficult and time consuming to measure the noise characteristics of MWs reliably in the past, our strategy can be a powerful tool and should be a significant breakthrough for the basic research and device applications of MW-based devices.In summary, we have developed a noise mapping strategy for the reliable identification and analysis of electrical noise in MW junctions. In this strategy, we patterned different MWs on the same gold substrate and placed a conductive AFM probe on the pattern to measure the electrical currents and noise PSD spectra simultaneously. By scanning the probe above the MW patterns during the measurement, maps of the 2 substrates using a thermal evaporation method. Typically, 10 nm of Ti was first deposited as an adhesion layer with a background pressure of approximately 3 × 10−6 Torr and a deposition rate of ~1 kÅ/s. Then, a 30 nm layer of gold was deposited with the same background pressure and a deposition rate of ~2 kÅ/s. The substances for molecular wires, including the mercaptopyridine and all alkanethiol molecules, were purchased from Aldrich. The SAM patterns of the molecular wires were prepared via the microcontact printing method as reported previously2 for 10 s. Then, the stamp was pressed down on a gold substrate for 3 s such that the molecules were transferred from the stamp to the substrate and formed the SAM patterns of the molecular wires. The SAM patterns of the multiple molecular wires, such as those in As a substrate, gold films were prepared on SiOA conductive AFM  in contact mode with a feedback loop was used with a conductive AFM probe based on Pt  to map the electrical currents and noise spectra of the SAM patterns of the molecular wires. During the measurement process, the probe first made direct contact on the SAM with a contact force of ~1 μN, and then, a bias voltage was applied between the probe and underlying gold substrate using a function generator . During the measurements, the contact force between the AFM probe and sample was maintained at 1 μN using the contact force feedback loop based on a position-sensitive photo detector in the AFM system. The electrical currents through the probe were converted to amplified voltage signals by a low-noise preamplifier , and the noise PSD at a specific frequency range was measured by a homemade spectrum analyzer. The homemade spectrum analyzer consists of a band-pass filter and an RMS-DC converter. We used the band-pass filter included in the preamplifier SR570 to measure the signal within the desired frequency range. We built the RMS-DC converter circuit using an AD737 chip . Finally, the noise PSD value at the central frequency of the measured frequency range was obtained by dividing the converted DC signals by the bandwidth of the used filter. The map of the electrical currents and noise PSDs was obtained by scanning the probe during the measurements with the AFM feedback on. The typical scanning speed was 0.5 μm/s. To achieve reliable mapping of PSD values at a specific frequency, the probe was held on each pixel of the sample for approximately 200 ms, and the data measured during the time period were averaged. For the measurement of the PSD spectra at different frequencies as shown in How to cite this article: Cho, D. et al. Direct mapping of electrical noise sources in molecular wire-based devices. Sci. Rep.7, 43411; doi: 10.1038/srep43411 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


To investigate the predictive factors for the development of Kaposi sarcoma-related immune reconstitution inflammatory syndrome (KS-IRIS) and long-term prognosis in patients starting combined antiretroviral therapy (cART).3 and a decrease of >1 log in viral-load once started cART. We compared individuals who met KS-IRIS criteria with those that did not and described the long-term follow-up.We studied a retrospective-cohort of consecutive antiretroviral-naïve patients with KS initiating cART from January 2005 to December 2011 and followed through June 2013. KS-IRIS was defined as ≥2 of the following: abrupt increase in number of KS lesions, appearance or exacerbation of lung-opacities or lymphedema, concomitantly with an increase in CD4+ cell-count ≥50 cells/mmp < 0.0001), eight died attributed to pulmonary-KS. Thrombocytopenia <100,000/mm3 at follow-up occurred in 36% of KS-IRIS vs. 4% in non-KS-IRIS patients (p = 0.0002), 45% KS-IRIS patients with thrombocytopenia died, non without KS-IRIS. Chemotherapy (bleomicyn–vincristine) was more frequently prescribed in KS-IRIS patients (88.6% vs. 29.6%) with no differences in outcome; 80% of all patients achieve KS complete remission, 52% of them never received chemotherapy. No difference between groups in the long-term follow-up (mean 52.4 ± 27.4 months) was found, only one patient developed a secondary malignancy (1.12%).We included 89 patients, 88 males; 35 (39%) developed KS-IRIS at a median of 10 weeks (IQR 4–16). KS-IRIS patients had more pulmonary-involvement  contains supplementary material, which is available to authorized users. Kaposi sarcoma (KS) was first described in the nineteenth century. In 1981 it became one of the signs that marked the beginning of the AIDS epidemic. This enigmatic disease is now recognized as a cytokine-mediated angioproliferative disease  caused bThe use of combined antiretroviral therapy (cART) has resulted in a dramatic reduction in opportunistic infections and mortality in patients with human immunodeficiency virus (HIV) infection , 8. The In the present study, we describe the prevalence and risk factors associated with the development of IRIS: clinical characteristics, type of therapy, outcome, markers of poor prognosis and long-term prognosis in patients with KS from a cohort of patients in Mexico initiating cART. We also estimated the incidence of secondary malignancies in long-term survivors.We performed a retrospective study of consecutive antiretroviral-naïve patients with KS who began cART between January 1, 2005 and December 31, 2011 at the National Cancer Institute of Mexico (INCan), a referral center for adult patients with AIDS-associated cancers. We included follow-up data up to June 30, 2013 provided the patients had >2 hospital-visits, and had not received systemic corticosteroids within the 2 months prior or concomitantly with the initiation of cART . We obtaWe included the following clinical features related with KS: presence and extent of mucosal involvement, number of skin lesions (considered disseminated if there were > 20 lesions or affecting ≥ 2 regions of the body), the presence and extent of lymphedema, gastrointestinal involvement diagnosed by endoscopy and confirmed by biopsy, and lung involvement . We further classified patients accordingly to ACTG staging classification , 24, and3 or ≥twofold rise in baseline CD4+ cell count associated with clinical abrupt worsening of previously existing KS  or the development of KS (“unmasked”) within the first 6 months after the initiation of cART . Seventy (81.5%) of the patients were in ACTG stage T1, with no difference among the stages between groups and in mortality (Table Combined ART was based on Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI) in 45 patients 51%), protease inhibitors (PI) in 43 patients (48%), and there was one patient with three Nucleoside Reverse Transcriptase Inhibitors (NRTI),  more common in the KS-IRIS group. Initial chest roentgenogram showed often disseminated reticular and nodular opacities, Gallium scans performed in 17 patients (14 with KS-IRIS and three with non-KS-IRIS) prompted by clinical symptoms and an abnormal chest X-ray were negative in nine and one respectively. Pleural effusion was also more frequent in patients in the KS-IRIS group compared to the non-KS-IRIS group .There was a significant difference between groups at baseline regarding the extension and number of KS lesions, as well as the presence of pulmonary involvement Table  more co3), which did not differ at baseline between groups, developed in 11 of 35 patients with KS-IRIS at the 12-week follow-up visit and almost half of them (45.5%) died, including five of the seven individuals with platelet counts <50,000/mm3. None of the patients in the non-KS-IRIS group had thrombocytopenia <50,000/mm3 at follow-up and only two (4%) had platelet counts between 50,000 and 100,000/mm3.The initial HIV viral load was significantly higher among patients with KS-IRIS (Table p < 0.0001), with a mean number of chemotherapy cycles of 3.9 ± 2.3 and 3.8 ± 2 cycles respectively, (p = 0.8), (Table Chemotherapy was administered to 31 (88.5%) individuals in the KS-IRIS group and to 17 (31%) in the non-KS-IRIS group (Sixteen patients 18%) died; eleven in the KS-IRIS group, eight of them were attributed to KS lung disease; with a median survival of 16 weeks (IQR 9‒50 weeks) after the initiation of cART. If patients survived the initial KS-IRIS phase and were adherent to cART, they had a good prognosis (p = 0.01). In the non- KS-IRIS group five patients died none related to KS, with a median survival time from cART initiation of 51 weeks (IQR 35–58) . Table Using Cox Proportional Hazard model the main predictor for the development of KS-IRIS was KS pulmonary involvement . Table The mean follow up of 81 patients surviving at least 26 weeks after cART initiation was 52.4 ± 27.4 months, for a total of 4.3 person/years for the whole group. Except for one case of Hodgkin’s lymphoma diagnosed 2 years after the diagnosis of KS, no other malignancies were diagnosed during follow-up. Incidence rate of secondary malignancy among patients with long-term follow up was 300 per 100,000 person-years.3, 75 patients (93%) had an undetectable HIV viral load. Five patients had viral replication ; two of them had abandoned cART and for one patient data was unavailable.Mean CD4 count at last visit was 400 ± 228 cells/mmThere were no differences between KS-IRIS and non-KS-IRIS patients in the prevalence of KS as a sole AIDS defining event or coexistence of other AIDS defining illnesses. KS-related mortality was significantly higher among patients with KS-IRIS, but it was not different for patients with one or more defining AIDS events Table .KS remains the most frequent AIDS associated malignancy in HIV-infected individuals seen at the National Institute of Cancer in Mexico, representing 41% of all initial visits to the AIDS Clinic during 2005–2011. In this study we describe the prevalence of KS-IRIS in a group of patients starting cART, Pulmonary involvement was associated to the risk of developing KS-IRIS, and thrombocytopenia at 12 week’s follow-up after starting cART was associated to death. Over a third of patients with KS achieve remission without chemotherapy solely treated with cART.The pathogenesis of KS is complex; it involves HHV-8/KSHV infection, cytokines and host-immune suppression , 28. HHVMSM with HIV or other active sexually transmitted diseases have the highest seropositive rates for HHV-8 . At our In 1997, the first case of KS exacerbation in a patient who developed KS-induced airway obstruction 4 weeks after initiating cART was described . To our Higher HIV viral load was associated with the development of KS-IRIS by univariate analysis, which is a finding that had previously been reported though not consistently . In two ACTG staging classification was not useful differentiating groups for IRIS-KS or mortality; we attribute this to the fact that most patients had extensive disease and low CD4.cART has dramatically reduced opportunistic infections and mortality of HIV patients in established market-economy countries where early HIV diagnosis is promoted with extended access to HIV treatment , 37. cARMycobacterium infection and cryptococcosis [Management of KS-IRIS has not been studied in controlled trials, but as opposed to some cases of coccosis , corticococcosis , 44, 45.coccosis .There are a wide variety of chemotherapy regimens published for KS –49. In tKS-IRIS is a time-limited phenomenon, but it can be a life-threatening one. Our experience shows that once the initial crisis is overcome, KS resolves in a high percentage of patients with limited or no chemotherapy and does not recur if patients maintain good adherence to cART.3) at 12 weeks of follow-up was significantly associated with patient demise.The overall prevalence of pulmonary involvement in our series was higher (35%) compared to other studies  , 50 likeThe overall incidence of secondary malignancies observed in this population is lower to what has been reported in patients receiving pegylated liposomal doxorubicin (PLD), 1.2% vs. 9.2% at a long-term follow-up, median time 48 months (IQR 33–80) for patient surviving >26 weeks, vs. 50 months (IQ-17–76) after last PLD dose, respectively . It is iA limitation of the study that can contribute to the high prevalence of KS-IRIS we found, is that our institution is a referral tertiary care oncological center and only patients with more extensive disease are referred to our institution, while patients with few lesions and no organ involvement stay in primary care units for HIV patients. Other limitation is the analysis was retrospective and diagnosing a patient with KS-IRIS was based on published criteria , 13 withAdditional information on preventing and treating KS-IRIS is warranted including optimal timing of cART initiation for patients who present with extensive skin lesions and lung involvement, the impact of HIV and HHV-8/KSHV viral loads and CD4/CD8 counts as well as the production of inflammatory cytokines in diverse clinical scenarios as well as the role that other co-infections may have on the development of KS . Anti-HHIt important to emphasize that abrupt clinical deterioration associated with IRIS does not represent cART failure; thus, a change of antiretroviral therapy is not warranted .In summary KS patients with pulmonary involvement at diagnosis had higher risk of developing KS-IRIS. Thrombocytopenia at 12-week follow-up was significantly associated with mortality as determined by multivariate analysis. Over a third of patients with KS achieve remission without chemotherapy. Individuals that survive the initial period of KS-IRIS and adhere to cART had a good long-term prognosis. The information of the causes of death are available as Additional file


Xenopus tadpoles. Dopamine (0.5–100 μM) reduced fictive swim bout occurrence and caused both spontaneous and evoked episodes to become shorter, slower and weaker. The D2-like receptor agonist quinpirole mimicked this repertoire of inhibitory effects on swimming, whilst the D4 receptor antagonist, L745,870, had the opposite effects. The dopamine reuptake inhibitor bupropion potently inhibited fictive swimming, demonstrating that dopamine constitutes an endogenous modulatory system. Both dopamine and quinpirole also inhibited swimming in spinalised preparations, suggesting spinally located dopamine receptors. Dopamine and quinpirole hyperpolarised identified rhythmically active spinal neurons, increased rheobase and reduced spike probability both during swimming and in response to current injection. The hyperpolarisation was TTX-resistant and was accompanied by decreased input resistance, suggesting that dopamine opens a K+ channel. The K+ channel blocker barium chloride  significantly occluded the hyperpolarisation. Overall, we show that endogenously released dopamine acts upon spinally located D2-like receptors, leading to a rapid inhibitory modulation of swimming via the opening of a K+ channel.Dopamine plays important roles in the development and modulation of motor control circuits. Here we show that dopamine exerts potent effects on the central pattern generator circuit controlling locomotory swimming in post-embryonic  Dopamine, for example, acts upon two principal classes of receptor which generally exert inhibitory  or excitatory  influences on neural circuitsNeural circuits of the spinal cord that generate rhythmic locomotor activity, so called central pattern generators (CPGs), are subject to profound modulatory influences which alter neuronal properties and synaptic strengths to confer flexibility on locomotor output and behaviourXenopus laevis tadpoles, dopamine has opposing, concentration-dependent effects on swimming: low dopamine concentrations activate high affinity D2-like receptors to inhibit spontaneous locomotor activity; while high dopamine concentrations facilitate locomotion by activating lower affinity D1-like receptorsDopaminergic fibres forming descending tracts from the brain offer a rich source of neuromodulatory input to the axial locomotor circuits in the spinal cords of aquatic vertebrates lamprey; amphibi510Xenopus development, around the time of hatching, when the animal possesses a simpler and much better defined spinal locomotor circuit+ channel.In the present study we have examined the role of dopamine at earlier stages of Xenopus tadpoles to assess its modulatory influence on spontaneous and electrically evoked episodes of fictive swimming. In keeping with a previous report on older free swimming Xenopus tadpolesIn an initial set of experiments, dopamine was bath-applied at a range of concentrations to immobilized Xenopus swim circuit are indeed mediated via actions at D2-like receptors, the broad-spectrum D2-like agonist quinpirole (25 μM) was bath applied. Quinpirole faithfully mimicked all of the effects of dopamine by rapidly reducing the occurrence of spontaneous episodes of fictive swimming and inhibiting the same parameters of motor output including cycle period, burst duration and burst amplitude  or excitatory  influences on neural circuits. To confirm that the inhibitory effects of dopamine on the mplitude . The facmplitude .nd post-otic intermyotomal cleft  was bath-applied . The timXenopus spinal cord where it acts, at least in part, via the activation of the D4 receptor.Next we wanted to test whether dopamine inhibition is constitutively active. Amongst the inhibitory D2-like receptors , the D4 receptor has been previously shown to play an especially important role in regulating spinal swim circuitry+ channel.Having established that dopamine acts via an intrinsically active pathway on D2-like receptors in the spinal cord to profoundly inhibit fictive swimming, we next wanted to explore the cellular mechanisms responsible for this network inhibition. Therefore, patch clamp recordings were made from spinal neurons that are rhythmically active during fictive swimming e.g., . The mos+ channel in spinal neurons, a range of pharmacological blockers of different K+ channel subtypes was applied in the presence of TTX to test which, if any, could occlude the subsequent quinpirole effect on membrane potential , a broad spectrum blocker of inward-rectifying K+ channels, significantly occluded quinpirole’s effect on membrane potential in a dose-dependent manner (+ channels (Kir1-7) is likely to be responsible for the hyperpolarising effects of dopamine on membrane potential. Within this family of K+ channels, previous studies have shown that G protein-coupled, inward rectifying K+ channels (GIRKs) are a common target for D2-like receptors1.1), but this failed to occlude the effects of quinpirole   with increasing amplitude (steps of 10 pA) was injected into neurons to assess any dopaminergic effects on their intrinsic excitability. In dopamine (50 μM), a larger amplitude current pulse was required to evoke an action potential . As in pXenopus laevis, dopamine has opposing actions depending upon which of these receptor classes is activated: low dose dopamine selectively activates higher affinity D2-like receptors to reduce spontaneous swim bout occurrence, while higher doses activate lower affinity D1 receptors to increase swimmingXenopus development when much more is known of the CPG circuit and when the tadpole is normally sessile until stimulated to swim; and secondly, what are the cellular consequences of dopamine receptor activation in this model system? Our extracellular ventral root recordings show that dopamine, acting solely via D2-like receptors at all concentrations tested, exerts strong inhibitory effects on the full range of locomotor parameters including the occurrence of spontaneous swimming, episode duration as well as the frequency, duration and amplitude of individual locomotor bursts , and glybenclamide (ATP-dependent K+ channel blocker) were unable to occlude the quinpirole hyperpolarisation. Therefore, with the current data we are unable to determine the exact K+ channel subtype involved. Although TEA had no significant effect on the hyperpolarisation of spinal neurons, it is possible that other BaCl-sensitive, TEA-insensitive potassium channels, such as flickering potassium channels+ -dependent potassium channelsXenopus swimming.Various lines of evidence from our intracellular recordings suggest that in on types  and 6. W+ channel shifts neurons away from firing threshold, and hence they showed reduced spiking during swimming and in response to current injection (A hyperpolarisation caused by the opening of a Knjection  and 8. TXenopus tadpoles is known to reduce PIR in the descending interneuron (dIN) population, also slowing the onset of the next bout of excitatory drive, slowing swim frequencyCan the effects of dopamine on overall network output be explained by the effects on the spiking properties of specific spinal neuron subtypes? In the lamprey, dopamine (10–100 μM) decreases the frequency of glutamate-induced fictive swimming through the D2 receptor-mediated depression of low-voltage activated (LVA) calcium channels on spinal commissural interneurons (cINs)42223Xenopus tadpolesXenopus motor behaviour may be mediated by an increase in D1 receptor expression. Although a systematic, developmental analysis of dopamine receptor expression is yet to be performed in Xenopus tadpoles, findings from other model systems support this overall developmental pattern of receptor expression. For example, a recent study in mice showed that high levels of inhibitory D2-like receptors are found in the spinal cord throughout development, whilst excitatory D1 receptor expression is low at early stages but increases with development, leading to an age-dependent increase in excitatory drive in the spinal cordXenopus tadpoles, the mechanism of excitation by D1 activation is also an avenue for future research. Interestingly, barium chloride depolarised spinal neurons by ~4 mV  of the University of St Andrews. All experiments were performed on newly hatched pre-feeding Xenopus tadpoles were immobilized by gashing the skin before placing in 12.5 μM α-bungarotoxin saline for approximately 30 minutes, and then mounted on a rotatable Sylgard platform in a bath of saline . Both sides of the trunk skin overlying the myotomal muscles were removed using a finely etched needle and forceps. The dorsal parts of approximately 7 rostral myotomes were freed from the spinal cord and the roof of the hindbrain and spinal cord was opened to the neurocoel to improve drug access and provide access for patch clamp electrodes. For whole-cell current clamp recordings, exposed neuronal somata were patch clamped using borosilicate glass pipettes (Harvard Apparatus Ltd) pulled on a Sutter P97 pipette puller. Patch pipettes were filled with 0.1% neurobiotin (Vector lab) in the intracellular solution  and had resistances of ~10 MΩ. Changes in membrane potential were recorded in current clamp mode using an Axoclamp 2B amplifier. Simultaneous extracellular recordings of fictive swimming were made with suction electrodes from ventral roots at intermyotomal clefts, and signals were amplified using differential AC amplifiers (A-M Systems Model 1700). Simultaneous intracellular and extracellular signals were digitized using a CED Power 1401, and displayed and stored on a PC computer using Spike 2 software. Fictive swimming was initiated by stimulating through a glass suction electrode placed on the tail skin, which delivered a 1 ms current pulse via a DS2A isolated stimulator (Digitimer).20 prior to the experiment (<1 hour) and stored on ice in the dark to minimise oxidation. All other drugs were made up to stock concentrations and frozen in aliquots, defrosted before an experiment, and diluted to final concentrations. Quinpirole hydrochloride (Tocris) and Bupoprion hydrochloride (Tocris) were both dissolved in distilled H20. L745,870 trichydrochloride (Tocris) was dissolved in DMSO.All drugs were obtained from Sigma-Aldrich or Tocris Bioscience and bath-applied to the preparation. Dopamine hydrochloride (Sigma) was always made up in distilled HElectrophysiological data were first analysed using dataview software  and all raw data were imported into Excel spreadsheets and analysed. Statistical analyses were conducted using PASW statistics 21. A rest time of 2 minutes was given between evoked episodes of swimming to ensure each episode was not influenced by preceding activity48Following patch-clamp recordings, animals were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, overnight in a refrigerator (∼4 °C). Animals were first rinsed with 0.1 M PBS , and washed in two changes of 1% Triton X-100 in PBS for 15 min with agitation. Next, animals were incubated in a 1:300 dilution of extravidin peroxidase conjugate in PBS containing 0.5% Triton X-100 for 2–3 hours with agitation, and washed again in at least four changes of PBS. Animals were then immersed in 0.08% diaminobenzidine in 0.1 M PBS (DAB solution) for 5 min, moved to a DAB solution with 0.075% hydrogen peroxide for 1–2 min, and then washed in running tap water. Finally, animals were dehydrated in 100% alcohol, cleared in methyl benzoate and xylene, and mounted whole between two coverslips using Depex. Neuronal cell bodies and axon processes were observed under a x40 objective to identify CPG neuron types. All reagents were obtained from Sigma or Tocris Bioscience.How to cite this article: Picton, L. D. and Sillar, K. T. Mechanisms underlying the endogenous dopaminergic inhibition of spinal locomotor circuit function in Xenopus tadpoles. Sci. Rep.6, 35749; doi: 10.1038/srep35749 (2016).


Vibrio vulnificus (V. vulnificus), a Gram-negative marine bacterium, can cause life-threatening primary septicemia, especially in patients with liver diseases. How V. vulnificus affects the liver and how it acts on macrophages are not well understood. In this report, we demonstrated that V. vulnificus infection causes a strong inflammatory response, marked expansion of liver-resident macrophages, and liver damage in mice. We demonstrated further that V. vulnificus activates mTOR in macrophages and inhibition of mTOR differentially regulates V. vulnificus induced inflammatory responses, suggesting the possibility of targeting mTOR as a strategy to modulate V. vulnificus induced inflammatory responses. Vibrio vulnificus (V. vulnificus) is a halophilic, Gram-negative and life-threatening marine bacterium [V. vulnificus infection can cause severe primary septicemia in patients with liver diseases such as viral hepatitis, advanced cirrhosis, and chronic alcoholic liver disease [V. vulnificus infection, mechanisms that regulate V. vulnificus-mediated responses remain unclear.acterium , 2. V. v disease . DespiteV. vulnificus can induce mTOR activation and how mTOR may regulate innate immune responses to this pathogen in macrophages is unknown.Upon infection, innate immune cells sense the pathogen and initiate cascades of events that result in the production of cytokines such as IL-1β, IL-6, and TNFα, which recruit other immune cells such as macrophages, neutrophils, and dendritic cells (DCs) to defend against pathogen invasion , 5. TollV. vulnificus could directly inflict liver damage and induce strong innate immune responses and Kupffer cell expansion. Moreover, V. vulnificus can activate mTOR in macrophages and inhibition of mTOR differentially regulates V. vulnificus induced inflammatory responses.In this report, we demonstrated that V. vulnificus infection. One of the liver’s key functions is to clear blood-borne pathogens and to prevent bacteria from spreading to other organs to cause sepsis [V. vulnificus have been found to cause systemic infection in mice [V. vulnificus 1.1758 strain can cause liver damage has been unclear. To examine whether V. vulnificus is capable of causing liver damage, we intraperitoneally (i.p.) injected V. vulnificus CGMCC1.1758 into wild-type C57BL/6J mice. Mice injected with 1×108 CFU V. vulnificus showed higher mortality than those injected with 5×107 CFU  and aspartate aminotransferase (AST) levels  levels . H&E sta) levels . Additioogenates . These oV. vulnificus infection, we performed immunofluorescence analysis of frozen liver sections of V. vulnificus-infected and control mice. As +CD11b+ Kupffer cells and CD11b+Ly6G+ neutrophils accumulated in the livers of infected mice. To further confirm Kupffer cell accumulation, we performed FACS analysis of liver mononuclear cells. As shown in +F4/80int Kupffer cells were considerably increased in V. vulnificus infected mice. Thus, acute V. vulnificus infection caused Kupffer cell accumulation in the liver.To determine the infiltrating cell types in the liver after V. vulnificus infection was associated with increased proliferation, we injected BrdU into mice immediately before V. vulnificus infection. Six hours after injection, Kupffer cells from V. vulnificus-infected livers incorporated more BrdU than those in uninfected mice, suggesting that these cells proliferated in infected mice  and CD98 (an amino acid transporter)  . In vitrand CD98 , suggestV. vulnificus induced inflammatory responses, we measured cytokines concentrations in the serum and liver homogenates after V. vulnificus infection using a multiplex flow assay. In V. vulnificus-infected mice, multiple cytokines, such as IL-27, IL-6, IL-10, and MCP-1, obviously increased in both blood and liver  or V. vulnificus lysates for 6 hours contained increased IKKα/β and IκBα phosphorylation (V. vulnificus was capable of triggering IKK-NFκB activation. Low dose (0.2 MOI) V. vulnificus treatment also induced phosphorylation of mTOR, S6K1, S6, 4EBP1 and Akt. However, 2 MOI of V. vulnificus-treated cells did not induce phosphorylation of these proteins  in vitro for 6 hours. As V. vulnificus induced IL-6, IL-10, TNF-α, and IL-1α production in these cells. In addition, live but not inactivated V. vulnificus induced GM-CSF, IFNβ, IL-27, and IL-1β production. Interestingly, treatment of BMMϕs with rapamycin during V. vulnificus stimulation resulted in decreased expression of GM-CSF, IFNβ, IL-27, and MCP-1 without obviously affecting the production of other cytokines. In rapamycin treated BMMϕs, V. vulnificus-induced S6K1 and S6 phosphorylation was abolished and Akt phosphorylation was deminished  that can induce mTOR activation, although these components and the pattern recognition receptors that lead to mTOR activation remain to be identified. As mentioned earlier, mTOR and its tight regulation play important roles in innate immune responses [V. vulnificus infection triggered strong inflammatory responses in vivo and V. vulnificus treatment induced production of multiple cytokines in macrophages. Among these cytokines, GM-CSF, IFNβ, IL-27, and IL-1β production but not IL-6, IL-10, TNF-α, and IL-1α production is dependent on mTOR activity. It would be interesting to further determine the underlying mechanisms by which mTOR controls production of selective cytokines during V. vulnificus infection.As tissue-resident macrophages, Kupffer cells are strategically located to clear bacteria in the liver during infection. Kupffer cells do not obviously proliferate in the steady state . We haveferation . Thus, Kesponses , 20–22. This study was carried out in strict accordance with the guidelines of the Zhejiang Provincial Animal Care and Use Administration Office (SYXK-ZJ-2005-0061). We performed all the animal experiments in accordance with protocols approved by the Wenzhou Medical University Animal Care and Use Committee (reference: wydw2014-0009).Vibiro vulnificus CGMCC 1.1758 strain, which we grew at 37°C in brain heart infusion broth (BHI) or on the BHI rabbit blood agar plate.We purchased C57BL/6J mice from Wenzhou Medical University Laboratory Animal Facility and were maintained under a SPF environment at the Central Animal Laboratory of Wenzhou Medical University. The mice were kept for 5–7 days to acclimatize after transport from the supplier. In the survival experiment, mice were observed every 30 minutes for 24 hours, and mice that survived for 24 hours were euthanized. L-929 cells were purchased from the Cell Bank of the Chinese Academy of Science in Shanghai and cultured them in RPMI1640 containing 10% heat-inactivated fetal bovine serum (Ausvin) and penicillin-streptomycin . The China General Microbiological Culture Collection Center provided the V. vulnificus. For survival experiments, we i.p. injected mice with 5×107 or 1×108 CFU of V. vulnificus suspended in 200 μl PBS or with PBS alone. We euthanized and exsanguinated the mice 6 hours after infection for blood and tissue collection. The liver was fixed it in 4% paraformaldehyde for histological analysis, and froze it in OCT medium (Thermo) for immunofluorescence analysis. To measure V. vulnificus CFU in the liver, we perfused infected mice with PBS before harvesting their livers. We cultured liver homogenate on BHI blood agar plates at 37°C for 24 hours. We measured serum AST and ALT levels using AST and ALT kits (NjjcBio) following the manufacturer’s protocol.We used i.p. injection to infect female C57BL/6J mice with V. vulnificus at the indicated multiplicity of infection (MOI) or V. vulnificus lysates to the cells. We collected supernatants at the indicated times for cytokine quantification. We then subjected the infected cells to lysate preparation and Western blot analysis by following a previous protocol [We isolated bone marrow cells and cultured them with 10% L-929 cell culture medium as described previously . We platprotocol . Anti-phWe measured multi-cytokines by using a LEGENDplex multi-analyte flow assay kit (13-plex) for mouse inflammation panel (Biolegend). We assessed the cytokine concentrations in sera, liver homogenates, and supernatants of cultured cells according to the manufacturer’s instructions. We collected data using a BD FACSAria II and analyzed them with LEGENDplex data analysis software (Biolegend).8 CFU of V. vulnificus two hours later. We harvested the liver from each infected mouse six hours after infection, isolating liver monocytes by following the previous protocol [g for 20 min at room temperature without break. Cells at the interface were collected, washed and resupended in IMDM (10% FBS) for further staining. We stained the cell surface with CD11b and F4/80 (Biolegend) and used a BrdU Flow Kit (BD Bioscience) for BrdU intracellular staining, following the manufacturer’s protocol.We intraperitoneally injected the mice with 1 mg BrdU (BD Bioscience) in 200 μl PBS, then with1×10protocol . BrieflyWe performed cell surface staining with fluorescence-conjugated antibodies in 2% FBS–PBS. We obtained fluorochrome-conjugated anti-CD11b, anti-F4/80, anti-CD71, and anti-CD98 from Biolegend. We identified cell death using a violet Live/Dead kit (Invitrogen). We performed intracellular staining for Alexa Fluor 647-conjugated p-Akt (Ser473) and p-4EBP1 (Thr37/46)  using BD Biosciences Cytofix/Cytoperm and Perm/Wash solutions. We collected data using a BD FACSAria II and analyzed them with FlowJo (Treestar).For histology analysis, we imbedded the fixed livers in paraffin, then cut thin sections, and following standard procedures, stained them with hematoxylin and eosin. We quantified the liver histological score of inflammation according to Ishak inflammation score , 44. Fort test and determined survival difference by log-rank survival analysis, performing all statistics using Graphpad Prism 5.0 software. P values are defined as follows: P < 0.05, P < 0.01, and P < 0.001.We analyzed statistical significance using the Student


Howeverem (SNS) , 12. ThiThe concept of leptin resistance is used to define states where hyperleptinemia is combined to lack of response to the hormone, with consequent maintenance of body weight excess and increased food consumption . Neverthvia activation of beta-adrenergic receptors post-synapticaly in target organs  and elevation of risk for developing cardiovascular diseases . MoreoveMost Obesities kNown Are Low In Sympathetic Activity). This hypothesis was built up from studies of SNS activity and norepinephrine decay in obese patients and rats (tabula rasa models precede the era of molecular genetics, which paved the way for molecular and functional diversity of seemingly alike cells. Indeed, the era of molecular genetics has enabled the mechanistic dissection of brain circuits, as well as, the immune system in spectacular ways. However, the molecular and circuit organization of the SNS, which innervates all known organs, is essentially unexplored. A molecular neuroanatomical map of the SNS may in turn revive the MONA LISA hypothesis. Once we understand the diversity of the SNS from a molecular and circuit standpoint, we then may be closer to resolving the mistery of leptin resistance.Taking into account all the aspects referred above, leptin resistance may also involve decreased sympathetic local activity, within the adipose organ. In this regard, and before the leptin era, George Bray and others proposed the “MONA LISA” hypothesis (and rats –46. Howeand rats . The conAll authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.