Our goal in the present study was to examine the effects of soluble oAβ on neuronal and synaptic structure in the amyloid precursor protein (APP) E693Q (“Dutch”) mouse model of AD, which develops intraneuronal accumulation of soluble oAβ with no detectable plaques in AD-relevant brain regions. We performed quantitative analyses of neuronal pathology, including dendrite morphology, spine density, and synapse ultrastructure in individual hippocampal CA1 neurons.Mounting evidence suggests that soluble oligomers of amyloid-β (oAβ) represent the pertinent synaptotoxic form of Aβ in sporadic Alzheimer’s disease (AD); however, the mechanistic links between oAβ and synaptic degeneration remain elusive. Most When assessing neuronal morphology and complexity we observed significant alterations in apical but not in basal dendritic arbor length in Dutch mice compared to wild type. Moreover, Dutch mice exhibited a significant decrease in dendritic arborization with a decrease in dendritic length and number of intersections at 120 μm and 150 μm from the soma, respectively. We next examined synaptic parameters and found that while there were no differences in overall synaptic structure, Dutch mice displayed a significant reduction in the post-synaptic density (PSD) length of synapses on mushroom spines, in comparison to wild type littermates.The structural alterations to individual neurons in Dutch mice along with the changes in larger dendritic spines support the Aβ oligomer hypothesis, which postulates that the early cognitive impairments that occur in AD are attributed to the accumulation of soluble oAβ first affecting at the synaptic level with subsequent structural disturbances and cellular degeneration.The online version of this article (doi:10.1186/1750-1326-9-41) contains supplementary material, which is available to authorized users. Soluble oAβ binds preferentially to synapses[in vivo; the injection of oAβ directly into the hippocampus of rats resulted in deficits in learning and memory[in vitro or in vivo in mouse models that possess soluble oAβ as well insoluble fAβ, and Aβ plaques  include amyloid-β (Aβ) plaque accumulation, neurofibrillary tangle formation, and synaptic and neuronal loss. How these factors ultimately contribute to memory loss and cognitive deficits that clinically characterize the disease remains unclear. In AD brains, Aβ can spontaneously aggregate and thereby assume a variety of conformational states ranging from monomers to soluble oligomers, protofibrils, and insoluble fibrils, which assemble to form extracellular plaques, 2. Furtsynapses, and addsynapses. This cod memory, 13. Whiues (see for reviAPP) are observed in patients with familial early-onset AD or dementia caused by cerebral amyloid angiopathy. Of the APP mutations causing AD or cerebral amyloid angiopathy, four occur at the E693 position of the protein, the Dutch (E693Q)[APP E693G (Arctic) or E693Δ variants show little or no fibrillar Aβ as detected by amyloid imaging[APP[APP located near the middle of the Aβ domain influence the propensity of Aβ to form oligomeric assemblies by disrupting the salt bridges on the protein that typically stabilize parallel β-pleated sheets and favor fibril and plaque formation, thereby promoting the formation and intraneuronal accumulation of oAβ[Mutations in amyloid precursor protein ( (E693Q), Arctic  (E693Q) and Ital (E693Q) mutation (E693Q). In cont imaging, 19. Curing[APP. Missensn of oAβ. Severe n of oAβ, 23. Thin of oAβ, 23. Recn of oAβ, indicatOur goal was to complement these behavioral findings by performing quantitative analyses of neuronal pathology, including dendrite morphology, as well as spine and synapse numbers in individual hippocampal CA1 neurons. These morphological features of the neuron are the site of critical memory forming processes–26. TwelBefore beginning the neuroanatomical comparisons in DU and WT mice, it was first necessary to confirm transgene expression and the presence of oAβ in the DU mice. Using the 6E10 anti-Aβ antibody we found that DU mice expressed amyloid and exhibited accumulation of intracellular Aβ  = 2.828; p = 0.013) but not in basal dendrites  = 0.857; p = 0.405) in DU mice compared to WT  = 6.31 p = 0.013) and intersections  = 0.82, p = 0.01) in DU dendrites compared to WT mice. These differences were most pronounced at a distance of 120 μm and 150 μm away from the soma, respectively. When basal dendrites were analyzed there were no significant differences observed for both basal dendritic length  = 0.01 p = 0.923) and basal intersections  = 6.31 p = 0.999) between DU and WT mice. Together these data suggest that soluble oAβ primarily affects the apical dendritic arbors, with no effect on basal dendrites of CA1 pyramidal neurons when it accumulates in situ. It remains to be assessed whether these effects are the result of decreased input to the CA1 from other affected hippocampal areas or a local effect in the CA1 region.We assessed whether CA1 neurons from DU mice would display differences in dendritic length and complexity compared to neurons from WT mice, and predicted that apical and basal dendrites from DU mice would be atrophic, and display perturbed arborization and complexity compared to WT mice. Representative examples of CA1 dendritic arbor reconstructions and dendrograms of WT and DU neurons are depicted in Figure t(8) = 1.967; p = 0.085). When we separated spines into apical and basal dendrites we observed a decrease in apical but not basal mushroom spine density (t(8) = 2.193; p = 0.06; and t(8) = 0.032; p = 0.975 respectively); however, as above, this did not reach significance. When the apical spine densities for each subtype were separated into proximal (50–100 μm from the cell soma) and distal (>100 μm from the cell soma), no differences were observed, and these data were therefore pooled.With the dendritic atrophy observed in apical dendrites in response to the accumulation of soluble oAβ, it is also possible that there may be alterations in dendritic spine density or the proportion of spine subtypes. We therefore analyzed WT and DU neurons for changes in the density of dendritic spines. We compared overall spine density of DU and WT mice, and also examined possible changes in thin, stubby, and mushroom spines, separately Figure . We obseNext we asked whether oAβ affects the density of perforated and non-perforated synapses on spines from both the stratum radiatum (SR) and the stratum lacunosum-moleculare (SLM) dendritic domains. Approximately 1500 synapses were reconstructed  across serial EM sections using the disector method. Analysis of the density of non-perforated and perforated synapses from SR and SLM revealed no significant difference between WT and DU mice Figure C, and tht(8) = 3.025; p = 0.016).We reasoned that there might be changes to the synaptic characteristics on specific spine subtypes. We chose to focus our investigation here on the mushroom spine subtype since this spine subtype is implicated in the formation and maintenance of memory, 29. AltAβ and its proposed neurotoxicity has been the focus of AD research since it was identified in the brains of AD patients three decades ago, 31. TheIn silico modeling of Aβ fibrillization[in vitro experiments[in vivo studies do report neuronal atrophy with proximity to fAβ in the brain and subsequent functional deficits (see[N-methyl-D-aspartate glutamate receptors (NMDARs) compared to large, mushroom spines that contain more 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptors (AMPARs), making synapses on mushroom spines functionally stronger[in vitro and ex vivo experimental models have reported direct binding of soluble Aβ to synaptic proteins at the PSD[In the current study, we have utilized a mouse model of AD that expresses the Dutch variant of APP. lization, cell-frlization and in veriments, 41 showeriments. In conteriments [in situ influences the proportion of silent synapses or dramatically alters the expression and distribution of synaptic receptors in the CA1. Additional behavioral and immunogold EM analyses in older DU mice (18 and 24 month-old) will also reveal whether their cognitive deficit and neuropathology worsens with age.The significant decrease we observed in PSD length of synapses only on apical mushroom spines may reflect disturbances in synaptic function and memory impairment in the presence of oAβ. A similar finding was reported by Nicholson et al. (2004) in their study of aged, learning-impaired Long Evans rats. These authors reported a significant (~30%) decrease in PSD length of CA1 SR synapses in the impaired group compared to control rats, and not(CaMKII). CaMKII (CaMKII), 70. A r(CaMKII). Furtherin vitro studies on the putative neurotoxicity of oAβ to dendrites, spines, and synapses that do report changes to neuronal morphology have used synthetic oAβ preparations that may not accurately match the toxicity profile of naturally occurring oAβ species occurring in vivo[in situ prior to this point, means that care should be taken in the interpretation of individual AD studies. Importantly, this also highlights the need for consistent methodological practices across AD research for improving and accelerating our understanding of the disease. We propose that the DU mouse model of AD represents a useful tool for the assessment of oAβ-mediated synaptic changes, based on our findings of detectable synaptic changes parallel to the onset of cognitive deficits in this model. Future studies using transgenic models expressing the Dutch, Arctic or E693Δ variants of APP and aimed at the development of imaging techniques to detect oAβ, in the absence of Aβ deposits would enable correlation of these synaptic changes with a potential clinical diagnostic tool.In the present study, we report that accumulation of soluble oAβ only in DU mice leads to discernible morphological changes to neurons, with an increase in dendritic atrophy accompanied by a decrease in PSD length that may be responsible for the cognitive impairment seen in these mice. Many  in vivo. In addi in vivo). Methodad libitum access to food and water. The DU mice used in the study contain the human APP gene harboring the Dutch mutation (APP E693Q) under control of the Thy1 promoter[42/Aβ40 ratio and exhibit plaque deposition, activated microglia, neuronal loss and cognitive deficits as early as 3 months of age[Male and female 12-month old mice were used in the study. All animals were group housed in micro-isolator cages under a 12-hour light/dark cycle and given promoter. These mpromoter. TgCRND8s of age–75. All Mice were anesthetized and perfused with 1% paraformaldehyde (PFA) followed by 4% PFA with 0.125% glutaraldehyde as described previously, 76. TheFree-floating hippocampal brain slices from DU and WT mice were processed for immunohistochemistry for Aβ using the 6E10 anti-Aβ antibody (Covance) as previously described, 77. TheFor intracellular injections, sections were incubated in 4’,6-diamidino-2-phenylindole  for 5 min to reveal the cytoarchitectural features of the pyramidal layer of CA1 of the hippocampus. The sections were then mounted on nitrocellulose paper and immersed in ice-cold 0.1 M PBS. Pyramidal neurons in the CA1 were subjected to an intracellular iontophoretic injection of 5% Lucifer Yellow (Molecular Probes) in distilled water under a direct current of 3–8 nA until dye had completely filled distal processes, 76, 78.To be included in the analysis, a loaded neuron had to satisfy the following criteria: (1) reside within the pyramidal layer of the CA1 as defined by cytoarchitectural characteristics; (2) demonstrate complete filling of dendritic tree, as evidenced by well-defined endings; and (3) demonstrate intact tertiary branches, with the exception of branches that extended beyond 50 μm in radial distance from the cell soma, 76, 78.Using an approach that precludes sampling bias of spines, CA1 dendritic segments were selected with a systematic-random method, 81. Denhttp://research.mssm.edu/cnic/) to examine global and local morphometric characteristics of dendrites and spines such as dendritic spine densities and dendritic spine shape. This software allows for automated digitization and reconstructions of neuronal morphology in 3D from multiple confocal stacks on a spatial scale and averts the subjective errors encountered during manual tracing using a Rayburst-based spine analysis[After deconvolution, confocal stacks were analyzed with NeuronStudio–84  encompassing the CA1 region of the hippocampus were prepared for EM as reported previously. Briefly3, and the height of the disector was 180 nm. Axospinous synapse density (per μm3) was calculated as the total number of counted synapses from both images divided by the total volume of the disector. The criteria for inclusion as an axospinous synapse included the presence of synaptic vesicles in the presynaptic terminal and a distinct asymmetric PSD separated by a clear synaptic cleft . Twelve sets of serial images across the same set of 5 consecutive ultrathin sections were taken from the SR and SLM of the hippocampus CA1 field and imported into Adobe Photoshop . To obtain a stereologically unbiased population of synapses for quantitative morphologic analysis, we used a disector approach on ultrathin sections as in previous reports, 88. Brit-test was performed. Sholl analysis was performed on apical and basal dendrites for total dendritic length, number of intersections and amount of dendritic material. Both Sholl and intersection analyses were analyzed using two‒way ANOVA followed by Bonferroni’s post‒hoc tests. To determine if the frequencies of different sized dendritic synapses differed between DU and WT mice, cumulative frequency curves of maximum spine head diameter and PSD length were examined using the Kolmogorov-Smirnov test. There were no statistical differences between male and female mice for these data, therefore sexes were grouped together. The statistical significance level for all data was set at an α level of 0.05 and all data were represented as mean ± SEM.All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software Inc). For neuron morphology and spine and synapse data, values for single cells were averaged for each animal and then per genotype and analyzed with GraphPad Prism 5. For the mean apical and basal dendritic length, and all spine and synapse data, a two-tailed Student’s (A) and PSD length (B) of synapses on CA1 hippocampal neurons in DU and WT mice. No shift in the distribution of head diameter or PSD length of CA1 synapses was observed in DU and WT mice. (TIFF 9 MB)Additional file 1: Figure S1: Cumulative frequency curves of synapse head diameter and PSD length. Cumulative frequency curves of synapse head diameter


However, a major impediment to the application of δ2H for inference of bat movements is that the relationship between δ2H of bat hair and precipitation tends to be species specific and is still unknown for some key species of conservation concern. We addressed this issue by using geo-referenced museum specimens to calibrate the relationship between δ2H of hair (δ2Hhair) and long-term δ2H of growing-season precipitation (δ2HGSprecip) at the site of collection for eastern red bats , one of the main species of bats experiencing large numbers of fatalities at wind-energy facilities in North America. Based on comparison of δ2Hhair and δ2HGSprecip values for males we estimated a period of molt of June 14–August 7. Within this period, male and female red bats exhibited a significant positive relationship between δ2Hhair and δ2HGSprecip. These results establish the relationship between δ2Hhair and δ2HGSprecip for red bats, which is necessary for the use of δ2Hhair to infer the movement and migration patterns of this important species. These results provide a critical resource to conservation biologists working to assess the impacts of environmental change on bat populations.Bats face numerous threats associated with global environmental change, including the rapid expansion of wind-energy facilities, emerging infectious disease, and habitat loss. An understanding of the movement and migration patterns of these highly dispersive animals would help reveal how spatially localized the impacts from these threats are likely to be on bat populations, thus aiding in their conservation. Stable hydrogen isotope ratios ( Bats living in temperate zones display a range of strategies for escaping unfavorable winter conditions. Species that survive through winter by hibernating in caves or buildings often have relatively sedentary populations or undertake regional migrations, whereas other species undertake seasonal migrations of hundreds or thousands of kilometers to find suitable winter habitat . Howeverδ2H), overcome these challenges and are a viable method to infer the summering grounds of bats  are compared with values of δ2H of growing-season precipitation (δ2HGSprecip) at the same locations. Prior studies have shown strong positive relationships between δ2Hhair and δ2HGSprecip for hoary bats  is among the bat species experiencing the highest levels of mortality at wind-energy facilities in the eastern United States  for red bat specimens that (1) had sufficiently detailed information to be able to geo-reference the location of collection, and (2) were collected during June–August. This period includes the time of year when red bat individuals are most likely to be resident on their summering grounds, as approximated from the estimated period of molt in hoary bats . Values of δ2H are expressed in parts per mil (‰) using the following equation: δ2H(‰) = , where R is the ratio of 2H/1H. δ2H sample data were normalized to the Vienna Standard Mean Ocean Water-Standard Light Antarctic Precipitation (VSMOW-SLAP) scale using a two-point normalization curve with USGS42 and USGS43, whose δ2H values of non-exchangeable hydrogen are −78.5 and −50.3‰, respectively. Most of the δ2Hhair values of the specimens were >−50.3‰, but prior studies suggest that linear extrapolation of normalization relationships for δ2H is appropriate for values within ∼100‰ of the range of the standards used for normalization  for each collection site. The small uncertainties associated with our approach for approximating the latitude, longitude and elevation of sample locations had little influence on the δ2H values of precipitation that were calculated for each site because δ2H values of precipitation exhibit greater variation across large than small environmental gradients . Specimen collection years spanned a period from 1900 to 2009. We subset samples by sex for initial analyses to assess potential intersex differences; specimens of unknown sex were excluded from these analyses.We used Google Earth to determine the approximate latitude, longitude and elevation of the collection location of each specimen, based on information provided on the specimen labels. Where information was restricted to broader geographic regions  we used values for a central point. Latitude, longitude and elevation values were entered in the Online Isotopes in Precipitation Calculator  of the difference between individual δ2Hhair and δ2HGSprecip values was minimized. To do this, we calculated the standard deviation of the difference between individual δ2Hhair and δ2HGSprecip values. We determined the presumed period of molt by visually identifying where the standard deviation was the lowest. We included samples collected during the presumed period of molt in subsequent reduced major axis (RMA) regressions. We performed RMA regressions to assess the relationship between δ2Hhair and δ2HGSprecip because of symmetry between the dependent and independent variables  suggest that this is the approximate period during which male red bats are typically resident on their summering grounds and synthesize new annual pelage. Males collected before June 14 or after August 7 were more likely to have molted at a location other than where they were collected. There was no clear trend in temporal variation of the standard deviation of δ2Hhair–δ2HGSprecip values for female red bats  for evaluation of the relationship between GSprecip . For malwas 33.0 . The stared bats .δ2Hhair values for male red bats exhibited a strong positive relationship with δ2HGSprecip during this period  and between July 1 and August 7  were stronger than the relationship between δ2Hhair and δ2HGSprecip between June 14 and August 7. When male and female bats (from June 14 to August 7) were combined, there was a strong positive relationship between δ2Hhair and δ2HGSprecip . The first, from δ2Hhair data from museum specimens and estimates of δ2HGSprecip from δ2Hhair data from δ2Hhair data were recalibrated to different standards and estimates of δ2HGSprecip were derived from the same model . The difference between the maps for red and hoary bats remained regardless of whether the To assess the species-specific nature of the relationship between e sample , which yme model  that we δ2Hhair and δ2H of precipitation. This relationship is often species-specific for different animals  at distant sites, such as at wind turbines or on their overwintering grounds.Stable isotope analysis has emerged as an important tool for studies of movement, migration, population connectivity, and habitat use of animals not amenable to traditional tracking methods . However animals , includi animals ; Table 1δ2Hhair and δ2HGSprecip values. Greater variation in δ2Hhair–δ2HGSprecip values for male red bats collected before June 14 and after August 7 suggests that individuals collected outside of the approximate timeframe of June 14–August 7 were less likely to have molted at the site of capture. This estimated period of molt is similar to the δ2H-inferred period of molt (June 20–August 23) reported for the hoary bat  compared to male red bats. The weaker relationship for females might be a function of delayed molt in reproductive females, as discussed above. Indeed, δ2HGSprecip had a stronger relationship with δ2Hhair for female red bats collected only in July and August than for females from June 14 to August 7. Although the precise timing of molt of female red bats warrants further study, the regression slopes and intercepts for males and females were not different  in the relationship between δ2Hhair and δ2HGSprecip for red bats.Within the estimated period of molt, we found significant positive relationships between ifferent  and ther dataset  were witδ2Hhair and δ2HGSprecip for male red bats. Although the precise reason for this discrepancy is uncertain, we offer two potential explanations. The δ2Hhair values from such bats would partly indicate their location the prior summer rather than of the year in which they were collected. A second possible explanation is that bats used in δ2HGSprecip during the last ∼100 years , whereas it may have a larger impact when using samples from a relatively small number of years .In contrast to our results, 00 years , there cδ2Hhair to identify the location of the summering grounds  of red bats of unknown geographic origin. In contrast to intraspecific similarities, our results underscore the species specificity of the δ2Hhair and δ2HGSprecip relationship, even among closely related bat species. For example, a δ2Hhair value of −40‰ yielded distinct δ2HGSprecip values and likelihood-of-origin maps for red and hoary bats based on using the regression presented here for red bats and those of δ2Hhair to infer the movement and migration patterns of red bats, and will enable future studies on red bat ecology and conservation, especially in the context of assessing the impacts of threats associated with global environmental change.Our results provide confidence for using 10.7717/peerj.629/supp-1Table S1Click here for additional data file.


Separation and analysis of biomolecules represent crucial processes for biological and biomedical engineering development; however, separation resolution and speed for biomolecules analysis still require improvements. To achieve separation and analysis of biomolecules in a short time, the use of highly-ordered nanostructures fabricated by top-down or bottom-up approaches have been proposed. Here, we reported on the use of three-dimensional (3D) nanowire structures embedded in microchannels fabricated by a bottom-up approach for ultrafast separation of small biomolecules, such as DNA, protein, and RNA molecules. The 3D nanowire structures could analyze a mixture of DNA molecules (50–1000 bp) within 50 s, a mixture of protein molecules (20–340 kDa) within 5 s, and a mixture of RNA molecules (100–1000 bases) within 25 s. And, we could observe the electrophoretic mobility difference of biomolecules as a function of molecular size in the 3D nanowire structures. Since the present methodology allows users to control the pore size of sieving materials by varying the number of cycles for nanowire growth, the 3D nanowire structures have a good potential for use as alternatives for other sieving materials. Since separation and analysis of biomolecules are essential processes, researchers spend much time carrying out these processes for clarification of biological activities of living things146789111213141516171819202122232425et al.Hind III in 600 s under an applied DC electric field, but it was still difficult to separate DNA molecules smaller than 1 kbp due to the fabricated nanopillar size and spacing limited by electron beam lithography process. Pennathur et al.et al.et al.To achieve separation and analysis of biomolecules in a short time, Volkmuth and Austin12et al.XhoI  under an applied DC electric field. Tabuchi et al.et al.On the other hand, nanostructures fabricated by bottom-up approaches have been suggested to realize separation and analysis of biomolecules in a short time. One example of a bottom-up nanostructures is self-assembled colloidal nanoparticles loaded into microchannels as artificial sieving matrices222324Here, we propose to use three-dimensional (3D) nanowire structures fabricated by a bottom-up approach and embedded in microchannels for the ultrafast separation of small biomolecules, such as DNA, protein, and RNA molecules. The proposed embedded nanowire structures have advantages of structures fabricated by both conventional bottom-up and the top-down approaches; in particular, relatively small structures (~10 nm) can be fabricated and the fabricated nanowire structures can be reused many times. Recently, we have reported a self-assembled nanowire array embedded in microchannels for single DNA molecule manipulation and fast separation of relatively large DNA molecules27812141621222324252 nanowires (10–20 nm in diameter) is shown in 2 nanowires backbone, which has a tetragonal rutile structure302 nanowire were growth on (001) plane, which has surface energy higher than (010) plane, and the orientation angle is about 90° b2[(2L/b)-1 + exp(2L/b)] where b is the Khun length of dsDNA and L is the DNA contour length) of 50–200 bp DNA molecules are approximately 24.9–59.5 nm, which is a comparable size to the pore size distribution in the 3D nanowire structures , protein A (45 kDa), streptavidin (52.8 kDa), β-galactosidase (116 kDa), and fibrinogen (340 kDa). Since the diametric dimensions of the protein molecules are very small (less than 100 nm) and their shapes are very complex, it is a challenge to fabricate structures with a pore size smaller than or similar to the protein molecule size in order to separate the non-denatured protein molecules. The separation resolution and the number of theoretical plates per column length for each protein molecule are summarized in 2 = 0.993), which confirmed that the migration behaviors of those proteins were by the Ogston sieving regimettransportation = d/μE, where d is pore size between nanowires, μ is electrophoretic mobility and E is applied electric field) was shorter than the diffusion time  of each protein molecule It should be noted that the 3D nanowire structures could separate DNA, protein, and RNA molecules in a short time. However, the exact separation mechanism of biomolecule in the 3D nanowires still difficult to explain due to the complexity of biomolecule migration behavior and the size of protein or RNA is very small for the individual observation, consequently theoretical and experimental works still require. One scenario to describes our achievement is a relatively large rigidity of the 3D nanowire structures (Young’s modulus ≈ 100 GPa)We have demonstrated that our proposed 3D nanowire structures embedded in microchannels as sieving materials provide fast separation and analysis of biomolecules. Our methodology allows users to control the pore size of the 3D nanowire structures by varying the number of nanowire growth cycles. The 3D nanowire structures showed capabilities to separate complex and very small biomolecules such as DNA, protein and RNA molecules in a short time due to the rigidity of the nanowire structures and their very small pore size which is difficult to attain by other fabrication approaches. The 3D nanowire structures provide an alternative approach to analysis of biomolecules and analytical time saving, consequently our methodology has good potential for commercial mass production and realization of reusable of bio-analytical devices.2O:Ce(NH4)2(NO3)6:HClO4, 85:10:5). After that, the microchannel was fabricated by RIE  under CF4 gas ambient. The microchannel depth was controlled to be 2 μm. Then, the inlet and outlet via holes (1.5 mm diameter) for the microfluidic system were drilled with an ultrasonic driller . We prepared metal catalysts to define the spatial position of the nanowires within the microchannel. A Cr layer (10 nm thick) was deposited within the microchannel. Positive resist  was coated on the microchannel by spin coating, and then the pattern was drawn by electron beam lithography . After developing the resist, the Cr layer pattern was removed with Cr etchant solution. DC sputtering was used to deposit an Au metal catalyst (particle size 3 nm) within the microchannel for nanowire growth. Then the resist was lifted off by sequential immersion in dimethylformamide, acetone and isopropanol. The SnO2 3D nanowire structures were grown on fused silica microchannel by the gold catalyst-assisted pulse laser deposition (PLD). Once the background pressure of the chamber was evacuated to be 5.0 × 10−6 Pa, oxygen and argon mixed gas (1:1000) was introduced into the chamber with the ambient total pressure of 10 Pa. Au film was preheated at the growth temperature 800 °C for 20 minutes then the ArF excimer laser was ablated to SnO2 target material for the nanowire growth. After 30 minute, the sample was cool down to room temperature for 30 minutes3637382 layer was deposited onto the SnO2 nanowires using the RF sputtering system to avoid charge interaction between nanowires and biomolecules. Finally, the microchannel was sealed by using a fused silica cover plate (130 μm thick) as described in the literature method131427Fabrications were carried out using the previously reported procedures27−4 to 10−6 M. Kim et al.The mixture of small DNA molecules, which consisted of 50, 100, 200, 300, 500 and 1000 bp molecules, was stained with YOYO-1 (Invitrogen) at a dye base ratio of 1:15 contained in concentrated buffer solution . The electrophoretic protein mobility measurements in the nanowire array structure were done by using trypsin inhibitor (Invitrogen), protein A (Invitrogen), streptavidin (Invitrogen), β-galactosidase (Novusbio), and fibrinogen (Invitrogen), stained with Alexa Fluor 488 at a dye molecule per mol ratio of 1:4, 1:5, 1:4, 1:3, and 1:8, respectively. We measured the fluorescence intensity of Alexa Fluor 488 dye molecule in each protein samples by microvolume (UV-VIS) spectrophotometer  as shown in An inverted fluorescent microscope  equipped with a high voltage sequencer  was used to apply the DC electric fields and a Hg lamp was used to observe fluorescently stained protein molecules. Fluorescence images were captured with an EB-CCD camera  through a 10 × /1.40 NA objective lens (Nikon). The images were recorded on a DV tape  and then analyzed by image-processing software .How to cite this article: Rahong, S. et al. Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules. Sci. Rep.5, 10584; doi: 10.1038/srep10584 (2015).


We demonstrated strong interactive environmental controls on emissions, calling for profound revision of emission algorithms.Isoprene emission is typically modelled using independent controls of light, temperature, and ambient [CO 2]. However, the isoprene emission rate is ultimately controlled by the pool size of its immediate substrate, dimethylallyl diphosphate (DMADP), and isoprene synthase activity, implying that the environmental controls might interact. In addition, acclimation to growth [CO2] can shift the share of the control by DMADP pool size and isoprene synthase activity, and thereby alter the environmental sensitivity. Environmental controls of isoprene emission were studied in hybrid aspen (Populus tremula × Populus tremuloides) saplings acclimated either to ambient [CO2] of 380 μmol mol–1 or elevated [CO2] of 780 μmol mol–1. The data demonstrated strong interactive effects of environmental drivers and growth [CO2] on isoprene emissions. Light enhancement of isoprene emission was the greatest at intermediate temperatures and was greater in elevated-[CO2]-grown plants, indicating greater enhancement of the DMADP supply. The optimum temperature for isoprene emission was higher at lower light, suggesting activation of alternative DMADP sinks at higher light. In addition, [CO2] inhibition of isoprene emission was lost at a higher temperature with particularly strong effects in elevated-[CO2]-grown plants. Nevertheless, DMADP pool size was still predicted to more strongly control isoprene emission at higher temperatures in elevated-[CO2]-grown plants. We argue that interactive environmental controls and acclimation to growth [CO2] should be incorporated in future isoprene emission models at the level of DMADP pool size.Plant isoprene emissions have been modelled assuming independent controls by light, temperature and atmospheric [CO Isoprene as a highly reactive and the most widespread volatile molecule emitted from a series of plant species plays a major role in air quality and climate, participating in ozone and secondary organic aerosol generation .2 concentration above approximately 100–150 μmol mol–1 inhibit isoprene emission  is catalysed by isoprene synthase . There is evidence that growth [CO2] can modify the instantaneous [CO2] response of isoprene emission and the maximum emission rate (2] (2] (i.e. the [CO2] at which the rate is measured) inhibited isoprene emission rate at temperatures of 30–35 ºC, but the [CO2] inhibition was lost at higher temperatures, indicating enhanced DMADP availability at higher [CO2]. Such an enhancement is consistent with the hypothesis that low DMADP availability at high [CO2] is associated with reduced chloroplastic inorganic phosphate levels due to imbalanced rates of starch and sucrose synthesis consuming triose phosphates and photosynthesis providing triose phosphates, ultimately leading to feedback inhibition of photosynthesis . We tested the hypotheses that elevated-[CO2]-grown plants would have modified environmental responses of isoprene emission and that such modified environmental responses in plants acclimated to different [CO2] would represent interactive controls on isoprene emission. In this model-based analysis, we integrated data reported in our previous studies  clone H200 (2] was maintained at an ambient level (average ± standard deviation) of 380±10 μmol mol–1 in chambers 1 and 3, and at an elevated level of 780±10 μmol mol–1 in chambers 2 and 4. The chamber air temperature was 28–30/23 °C (day/night), relative humidity was 60%, and light intensity at the top of the plants was 500–800 μmol m–2 s–1 for the 12h light period, resulting in a moderately high daily integrated growth light of 28.1mol m–2 d–1  .In this study, we included data for four series of replicate experiments reported by –2 s–1, and relative air humidity of 60%). Once the steady-state gas-exchange and isoprene emission rates had been established, the temperature responses of photosynthesis and isoprene emission were measured at a moderately high light intensity of 500 μmol m–2 s–1 (growth light intensity) and a strong light intensity of 2000 μmol m–2 s–1 at the [CO2] of 380 and 780 μmol mol–1. During the measurements, the leaf temperature was increased in steps of 5 °C from 30 to 50 °C, and the values of the isoprene emission rate were recorded for 8min after the change in temperature (a) such that steady-state photosynthesis and isoprene emission rates cannot be reached at these higher temperatures.Isoprene emission measurements were conducted after 30–40 d of growth under the given conditions when the plants had filled the chambers using individual attached fully mature leaves as described in detail by perature  and, altperature  and isopRL, as:To normalize the environmental responses of isoprene emission, we calculated the relative light-dependent increase of isoprene emission, I2000 is the isoprene emission rate at the light intensity of 2000 μmol m–2 s–1 and I500 is that at 500 μmol m–2 s–1. Analogously, the relative temperature-dependent change in isoprene emission (RT) was calculated as:where I30 is the emission rate at 30 ºC and IT is that at temperature T.where The temperature response of isoprene emission rate was also fitted by an exponential function with a maximum :T is the leaf temperature in K, R (8.314 J mol–1 K–1) is the gas constant, c is the scaling factor, ΔHa (J mol–1) is the activation energy, ΔHd (J mol–1) is the deactivation energy, and ΔS (J mol–1 K–1) is the entropy term. The explained variance of temperature relationships (r2) was in all cases >0.98. From this equation, the optimum temperature for I, Topt .To characterize the initial increase of isoprene emission rate with increasing temperature, we also calculated the average value of QCDMADP, nmol m–2) and on the isoprene synthase rate constant :The temperature response of isoprene emission is a mixed response that is driven by temperature effects on the DMADP pool size . Using available information on k and CDMADP at 30 °C , the response coefficients were only employed to gain insight into the changes in the light sensitivity of isoprene emission.Implicit in Eq. 5 is that the analysis  that pro2] treatments (380 vs 780 μmol mol–1) are denoted as ‘ambient’ and ‘elevated’, and the measurement [CO2] (380 vs 780 μmol mol–1) as 380 and 780. Thus, in this analysis, we had four combinations of growth and measurement [CO2]: ambient (380), ambient (780), elevated (380), and elevated (780), and two additional combinations of the measurement light intensity: a moderately high light intensity of 500 μmol m–2 s–1 and strong light intensity of 2000 μmol m–2 s–1. The effects of combinations of [CO2] treatment and measurement [CO2] at different light intensities and temperatures were analysed by analysis of variance (ANOVA) followed by Tukey’s test (growth [CO2] treatments involving independent samples) and by paired-samples t-tests (paired comparisons between different light and measurement [CO2]). Correlative relationships among leaf traits were analysed by linear regressions. To compare the statistical relationships among [CO2] treatments at different light intensities and measurement [CO2], analysis of covariace (ANCOVA) was used. The separate slope ANCOVA model with the interaction term (treatment with covariate) was fitted first, followed by the common-slope model (without the interaction term) when the interaction term was statistically not significant. For all analyses, we used SPSS 17.0 (IBM SPSS Statistics), and all statistical tests were considered significant at P<0.05.In the following, the growth [COI) up to 45–50 ºC  for isoprene emission did not depend on the measurement [CO2] for ambient-[CO2]-grown plants, but Topt was greater at the measurement [CO2] of 780 μmol mol–1 than at 380 μmol mol–1 in elevated-[CO2]-grown plants  and with traits characterizing the temperature sensitivity of emissions to lower and higher temperatures . As isoprene synthase itself has a very high optimum temperature of around 50 ºC  (P>0.1), RT×(light intensity) (P>0.6) and RT×(measurement [CO2]) (P>0.7) were not statistically significant. According to the common-slope ANCOVA model, both light intensity , and the correlations were much weaker for RT values calculated for temperatures of 45 °C  and 40 °C . In addition, differences in Topt were mainly associated with differences in the deactivation energy . Thus, the magnitude of the initial increase of isoprene emission at lower temperatures and the onset of the emission decrease at higher temperatures were essentially independent.In contrast to these correlations, k) and with the DMADP pool size  and I versus CDMADP were not significantly different among elevated- and ambient-[CO2]-grown plants. However, elevated-[CO2]-grown plants had a lower isoprene emission rate at a given k and higher isoprene emission rate at a given DMADP pool size .The isoprene emission rate through the temperature range 30–50 °C increased both with the predicted isoprene synthase rate constant .The light sensitivity of isoprene emission was positively correlated with the DMADP response coefficient across all the data, while the correlation was negative for the response coefficient for 2] treatments on leaf isoprene emission, identifying three novel features of how isoprene emissions respond to light and temperature in plants grown at different [CO2]:Our study highlights a complex interplay between different environmental drivers and growth [CO10) for isoprene emission varied in dependence on light intensity and growth [CO2] /I30 (I50 – I30)/I30, itself depends both on temperature effects on isoprene synthase activity and DMADP pool size, but provided Topt is less than the optimum for isoprene synthase activity, the way this characteristic is correlated with Topt depends on the extent to which isoprene emission is controlled by the DMADP pool size at higher temperatures. Thus, a greater Topt at a given value of (I50 – I30)/I30 in ambient-[CO2]-grown plants (What could be the mechanism for light- and [CO30)/I30 . The temn plants  is in ag2] of 780 μmol mol–1 had both a greater Topt (I50 – I30)/I30  suggested that isoprene synthase limited the flux at higher temperatures less in elevated-[CO2]-grown plants than in ambient-[CO2]-grown plants (data not shown). We argue that additional studies are needed that explicitly characterize the isoprene synthase temperature dependencies in plants grown under different [CO2] conditions.Leaves grown and measured at the higher -grown plants measured at 780 μmol mol–1 and for elevated-[CO2]-grown plants measured at 380 μmol mol–1  and moderate  leaf temperatures.Differences in average Qol mol–1  further nt light  is indicer Topt  suggests2]-grown plants is in agreement with experimental observations on their lower DMADP pool size and greater isoprene synthase activity. Given the smaller DMADP pool size, which strongly curbs isoprene emission, any increase in DMADP pool size at higher light readily results in a higher isoprene synthesis rate , a larger pool of DMADP relative to isoprene synthase activity can result in an increasingly non-linear Michaelis–Menten-type hyperbolic response , reducing the increase of isoprene emission for a given increase of DMADP pool size.The enhanced light sensitivity of isoprene emission in elevated-[COsis rate . This rerate (2] , possibl2]-grown plants under high measurement [CO2] was maintained over the entire temperature range. We suggest that these patterns result from multiple mechanisms operating at different parts of the temperature response of light sensitivity. First, the increase in temperature is initially associated with enhanced DMADP synthesis rate (Although the light enhancement of isoprene emission became weaker with increasing temperature, the stronger light enhancement in elevated-[CO2]-grown leaves under high measurement [CO2] and high temperature (2]-grown leaves under high measurement [CO2]. However, heat-depressed quantum yield of photosynthesis and photosynthetic electron transport as observed by 2]-grown plants. This, combined with the lower contribution of alterative carbon sources as (see Given these modifications, it is still puzzling why the light sensitivity of isoprene emission remained greater in elevated-[COperature . This res as see .2] treatments and under different measurement [CO2] and light intensities that collectively suggest that the effects of environmental drivers interactively affect isoprene emission at the level of the DMADP pool size. Thus, future models should focus on predicting integrated environmental controls on DMADP pool size rather than considering each environmental driver independently of others. Several semi-mechanistic models have recently been put forward that link isoprene emissions to photosynthetic electron flow and isoprene synthase activity (Our study highlights a number of important differences among temperature responses under different growth [CO2] on isoprene light and temperature responses. Consideration of such effects in models again requires understanding of growth [CO2] effects on isoprene synthase activity, changes in DMADP partitioning between isoprene synthesis and larger molecular mass isoprenoids, and possible modifications in isoprene synthase temperature responses. Process-based simulation of the competition for DMADP by isoprene synthase and geranyl diphosphate synthase might be particularly difficult, although linking GDP synthesis to carotenoid turnover rate as driven by photo-inhibition and heat stresses (2] (2] regimes is needed to gain insight into the factors controlling the partitioning of DMADP among isoprene and other competing pathways. Such an understanding is crucial for realistic parameterization of interactive environmental control of isoprene emission under global change.The study further highlights the important interactive effects of acclimation to growth [COstresses  can be a


TiO2 NRs/NCQDs nanohybrids were prepared by controlling the experimental condition. TiO2 NRs were self-assembled to form the nanopores with good photocurrent conversion efficiency. NCQDs possessed carboxyl groups (−COOH) and amino groups (−NH2) in the preparation process. −COOH and −NH2 groups played important roles for anchoring the capture probes (5′ primer and 3′ primer) through covalent binding. The ultrasensitive and stable detection for TXNDC5 was achieved by the specific recognition between the capture probes and the targets. The fabricated aptsensor showed excellent performance with a wide linear range (0.5 fmol/L ∼ 10 nmol/L) and a low detection limit of 0.1 fmol/L. This kind of aptsensor would provide a potential application for TXNDC5.The over expression of thioredoxin domain-containing protein 5 (TXNDC5) can promote the growth of castration-resistant prostate cancer (CRPC). A novel highly sensitive photoelectrochemical (PEC) aptsensor was developed for the detection of TXNDC5 by using the nanohybrids (TiO Prostate cancer (PCa) is the second leading malignancy in men worldwide in recent years and the incidence has been increasing continuously4568910111214151617Compared with the traditional inorganic semiconductor QDs, CQDs have multiple excellent performances1920232 is one of the most studied semiconductor nanostructures26272 strongly depends on the dimensionality. Highly oriented TiO2 single-crystalline NRs grown on the transparent conductive substrates have been considered as the optimum choice of the solar cell materials2 NRs is its ineffective utilization of the visible light as the irradiation source. Bandgap engineering by possible modification of TiO2-based materials is one of the plausible approaches to enhance the performance of TiO2 NRs, such as QD-sensitized.TiO2 NRs/NCQDsnanohybrids as the PEC signal medium was constructed  was used to reveal the small size of NCQDs (2/NCQDs nanohybirds. TiO2 NRs and as-assemble microsphere was not rigid and somewhat powder-like attachment can be distinguished on the surface. This suggested that NCQDs hybridized successfully with TiO2 NRs. EDS result in 2NRs and NCQDs.Scanning electron microscope (SEM)images in of NCQDs . HRTEM i−1. The peak at 1224 cm−1 indicated the characteristic stretch of C-N bond. C=O bending vibration was at 1390 cm−1. The peak of 1625 cm−1 was associated with stretching vibrations of C-O band. The peak at 1730 cm−1 meant C=O stretching vibration. The weak peaks in the range of 2820–3050 cm−1 were related to C-H bond stretching vibrations. A broad peak centered at 3440 cm−1 suggested the presence of many hydroxyl groups on the NCQDs surfaces. All the above mentioned indicated that NCQDs had abundant carboxyl and amino groups on their surfaces. The doped amino and carboxyl groups of NCQDs can increase the hydrophilicity and the combination ability with the detection targets in aqueous systemsd of 0.385 nm, which agreed with the (002) lattice spacing of carbon-based materials with turbostratic disorderd value was larger than that of graphite (0.34 nm), which indicated that NCQDs possessed abundant oxygen-containing functional groupsd value was same to the carbon nanoparticles from pyrolyzing ethanolamineaphite 0. nm, whic2 NRs, TiO2/NCQDs. Rutile phase TiO2 was produced and it can be confirmed by XRD pattern. For TiO2 NRs, seven diffraction peaks marked (1–7) can be respectively indexed to the (110), (101), (111), (210), (211), (220) and (002) plane. The addition of NCQDs had no obvious influence on the crystallinity and the phase purity of the resultant products. No diffraction peak resulted from NCQDs can be found, which may be attributed to the poor crystallinity and low content2 NRs, TiO2/NCQDs under the visible light radiation. TiO2 could be only effectively activated under the light with short wavelength. The viewpoint was proved by the relatively weak photocurrent response even in the presence of AA in 2/NCQDs were apparently increased about 20 times (b), which proved the addition of NCQDs enhanced PEC behavior. This might be explained that h+/e− pair separation efficiency was distinctly enhanced through the electron injection between NCQDs and TiO2 NRs.2 NRs, NCQDs, TiO2/NCQDs, UV-vis absorption spectra were studied , which suggested that the composition can enhance the visible light absorbancy for both TiO2 and NCQDs.To further explore the PEC properties of TiO studied . Curve a+/e− pairs. It can provide the proof for the photogenerated h+/e− pair recombination process from another aspect2 NRs, which was obvious stronger than those of NCQDs (curve b) and TiO2 NRs/NCQDs (curve c). It indicated a higher recombination efficiency of the photogenerated h+/e− pairs and a lower efficient electron injection from NCQDs to TiO2. PL intensity of NCQDs was lower than that of TiO2, which suggested that NCQDs owned better light absorbancy and photocurrent conversion efficiency. And also the addition of NCQDs into the nanohybrids decreased PL intensity which illustrated that NCQDs improved the photoelectrons transport and benefited the photoelectrons injection from occupied valence band (VB) to conduction band (CB). Therefore, NCQDs can enhance the PEC property of the wide band-gap TiO2 although the emission peak showed a blue shift.PL was a persuasive tool to illustrate the separation and recombination efficiency of photogenerated h−) was excited from VB to CB and the hole (h+) was formed in the VB due to the presence of a band gap under the light irradiation. However, the recombination of the electron and the hole took place generally. For TiO2, the photogenerated electrons more efficiently transferred to CB of TiO2 under the light irradiation. But subsequently the electrons were transferred directly into the VB of TiO2 NRs without a transition through the excited state392 to produce the e−/h+ pairs. However, when NCQDs were introduced into the composites system (TiO2/NCQDs), a mass of the visible light was converted to a shorter wavelength, which may increase the photogenerated e−/h+ pairs to enhance the PEC activity.The PEC performance mechanism proposition was shown in 2 and NCQDs were shown in 2 NRs was 3.2 eVvs. normal hydrogen electrode, NHE)2 NRs can be deduced about 2.76 V (according to Eg = VVB–VCB). The band gap of NCQDs was about 1.9 eV442 NRs to produce the e−/h+ pairs. NCQDs can accept the photogenerated electrons from TiO2 NRs and promote the separation of the photogenerated e−/h+ pairs.The relative Femi level alignment of rutile TiO2 NRs. Under the visible light irradiation, the electrons in the VB of NCQDs obtained enough energy and injected to the CB. The photogenerated electrons can be easily injected from NCQDs into TiO2 NRs via the interface injection. However, the holes on the VB of TiO2 NR scan transfer to NCQDs. As a reducing agent, AA acted as an electron donor to trap the holes in the VB of TiO2, which inhibited the e−/h+ recombination and improved the photocurrent responseThus, from the schematic diagram of the process, it can be seen that the VB and CB energy levels of NCQDs lied above those of TiO2 NRs and the electrons can be shuttled freely along the conducting paths in NCQDs. The combination of TiO2 NRs/NCQDs benefited for the charge separation and for hindering e−/h+ recombination472/NCQDs. N-doping can lower the work function of carbon nanomaterials and the lower work function of NCQDs produced much smaller barrier between NCQDs and TiO2. The inference was also proved by UV-vis and PL measurement as shown in −/h+ pairs.Therefore, NCQDs in the nanocomposites facilitated the transfer of the electrons from TiO2 NRs, the composition methods (three methods) of TiO2 NRs and NCQDs, the application amount of NCQDs composited with TiO2 NRs, pH values and the application amount of 3′ and 5′ primers were investigated in this study . This indicated that the composition of NCQDs with TiO2 NRs conspicuously increased the photocurrent. Because of the composition of NCQDs and TiO2 NRs, the electron transfer efficiency enhanced much and the recombination probability between the electrons and the holes was depressed apparently. And also the composition manner was also investigated . The bases of the primers owned−NH2 groups and TiO2/NCQDs have −COOH groups. When the primers were incubated on the modified ITO electrode surface, the interaction between the amino groups of primers and the carboxyl group of TiO2/NCQDs would occur under the activation of EDC/NHS amidization protocol. TiO2/NCQDs can immobilize the primers probes on ITO electrode through the interaction. But the bases can not transfer the electrons and absorb the visible light, which would result in the obstruction of the absorption of the light and the suppression of the electrons transfer. This would cause the decrease of the photocurrent and the experimental results proved this speculation. Therefore, the PEC aptsensor was fabricated as expected.The fabrication procedure of the PEC aptsensor could also be monitored by the photocurrent responses under the optimal experimental condition . PB solustigated . TiO2/NCI (μA) = 2.901 + 0.901 lgcTXNDC5 (pmol/L) and the correlation coefficient was 0.991. A detection limit of 0.1 fmol/L was obtained for the reported aptsensor. The proposed PEC aptsensor therefore showed a wide linear range and a low detection limit for the determination of TXNDC5. The low detection limit may be ascribed to good separated excitation energy of TiO2/NCQDs and the specific recognition between the capture probe and the target.In order to investigate the possibility of the aptsensor applied for TXNDC5 analysis, quantitative detection of TXNDC5 was operated under the optimal conditions. The process began after incubating various concentrations of TXNDC5 on ITO electrode. The detection results were given in Stability and reproducibility were two important parameters affecting the practical application of an aptsensor. 2 NRs/NCQDs nanohybrids expressed significantly enhanced PEC property. The combination of NCQDs improved the charge separation efficiency and the charge transfer ability, and suppressed the h+/e− recombination effectively. The developed aptsensor displayed the ultra-sensitivity and good stability. Thus, it provided good detection effect for TXNDC5 and might provide a feasible platform to determinate of other analyses.This work proposed a novel PEC aptsensor for the rapid and ultrasensitive detection of TXNDC5. The specific detection was realized by specific recognization between the capture probe and the target. When exposed to the visible light, the signal generator of TiO2PO4 and 0.067 mol/L Na2HPO4) were used as the electrolyte for all electrochemistry measurements. All other chemical reagents were analytical reagent grade and directly used without further purification. The ultrapure water (resistivity of 18.25 MΩ∙cm) and pipette tips were put into a LDZX-30KBS pressure steam sterilizer  and sterilized at 121 °C for 40 min. After cooling to room temperature, the tips were stored in a 4 °C refrigerator.Phosphate buffered solution . HRTEM image was recorded by JEM-2100F microscope . FT-IR spectra were obtained with Perkin-Elmer 580B spectrophotometer . XRD patterns were collected from D8 focus diffractometer . PEC tests were carried out on electrochemical work station . UV-vis measurements were obtained on Lambda 35 UV-vis spectrometer . PL spectra were obtained on LS-45/55 PL spectrometer .TXNDC5 was prepared according to our previous work2 NRs were prepared according to the method in the literature with slight modifications3 aqueous solution and 4 g of NaCl were added into 10 mL of distilled water under stirring. The solution was put into a Teflon-lined stainless steel autoclave with a capacity of 50 mL. The autoclave was sealed and heated at 100 °C for 12 h. After cooling to room temperature, the products were washed with distilled water and absolute ethanol for several times. Then the products dried under vacuum for use.TiONCQDs were prepared as the method described in the literature with slight modifications2 NRs/NCQDs nanohybrids were prepared as described in the literature with slight modifications3 aqueous solution, 4 g of NaCl and 10 ~ 28 mg of NCQDs powder were added into 10 mL of distilled water under stirring. The following procedure was same to the preparation of TiO2 NRs. TiO2 NRs/NCQDs nanohybrids were prepared as method 2, 3 (ESI†).Method 1:TiO2/NCQDs nanohybrids were dropped on ITO electrode and dried in a 4 °C refrigerator. Thirdly, 15 μL of diluted 5′ primer was dropped on TiO2/NCQDs/ITO electrode surface using an EDC/NHS amidization protocol and incubated in a 4 °C refrigerator for 4 h. After that, 15 μL of 3′ primer was dropped on 5′ primer/TiO2/NCQDs/ITO electrode using an EDC/NHS amidization protocol and incubated in a 4 °C refrigerator for 4 h. At last, TXNDC5 ssDNA with different concentrations were dropped on 3′primer/5′primer/TiO2/NCQDs/ITO electrode surface respectively and incubated at 4 °C for 4 h.The fabrication procedure of the PEC aptsensor was shown in The PEC measurements were performed immediately after the incubation of TXNDC5 ssDNA. A conventional three-electrode system was used in all the PEC experiments. A platinum wire was the auxiliary electrode. A KCl-saturated calomel electrode (SCE) was the reference electrode. The modified ITO electrode was used as the working electrode. A commercial LED light (430 nm) was used as the irradiation energy in all the PEC tests. PB containing 0.1 mol/L of ascorbic acid (AA) was used as an electrolyte solution for all the PEC measurements. The bias voltage was 0.1 V. Both light duration and no light duration were 20 s. All the PEC experiments were operated at room temperature.How to cite this article: Pang, X. et al. Enhanced photoelectrochemical aptasensing platform for TXNDC5 gene based on exciton energy transfer between NCQDs and TiO2 nanorods. Sci. Rep.6, 19202; doi: 10.1038/srep19202 (2016).


By tuning the etching depth and period of the antidot lattices, the band folding caused by the antidot potential leads to the formation of minibands and band inversions between neighboring subbands. The intersubband spin-orbit interaction opens considerably large nontrivial minigaps and leads to many pairs of helical edge states in these gaps.Based on the Born-Oppemheimer approximation, we divide the total electron Hamiltonian in a spin-orbit coupled system into the slow orbital motion and the fast interband transition processes. We find that the fast motion induces a gauge field on the slow orbital motion, perpendicular to the electron momentum, inducing a topological phase. From this general designing principle, we present a theory for generating artificial gauge field and topological phase in a conventional two-dimensional electron gas embedded in parabolically graded GaAs/In Exploring of various topological quantum states is always one of the central issue of condensed matter physics124567891011121314151617181920212223242578xGax1−As/GaAs two-dimensional electron gas (2DEG) with antidot lattices can be driven into the TI phase. The 2DEGs provide a promising playground for realizing TI states with quite large nontrivial gap (~20 meV) operating at liquid nitrigen temperature regime, instead of searching new materials containing heavy atoms. We first present a general analysis for generating an artificial gauge field in a semiconductor 2DEG, then we demonstrate band inversion between neighboring subbands because of inter-subbands spin-orbit interaction (ISOI) utilizing antidot lattices created by well-developed semiconductor etching technique, and generate the TI phase with many pairs of helical edge states. This suggests a completely new method to generate topological phase in conventional semiconductor 2DEGs without strong spin-orbit interaction (SOI), at liquid nitrigen temperature regime.In this work, we demonstrate that conventional semiconductor GaAs/Inτ, and taking d3 = 0, we get the Hamiltonian for a 2DEG with Rashba and Dresselhaus SOIs, where α and β are the strengths of Rashba and Dresselhaus SOIs, respectively. Next, we divide the total Hamiltonian at the band edge into the intra-band , slow part 2 meV), fast part HIB and First we discuss the emergence of an artificial gauge field in a system of electrons in a 2D system described by a low-energy single-particle Hamiltonian An leads to an effective Lorentz force E:where sn = ±1 denotes spin up or down state while tn = ±1 denotes the conduction or valence band, respectively . The Chern number Fxy,n in the Brillouin zone. The sign change in M would induce a change of the Chern number by 1, which corresponds to the topological phase transitionwhere τ. In order to generate the gauge field and realize TI phases in a 2DEG, one needs to create minibands and band inversion in 2DEGs. Based on the above designing principle, we will create topological phase in conventional semiconductor 2DEG. This is the first demonstration of the formation of a TI phase in the s-like band systems, i.e., a 2DEG with nanostructured antidot lattice shown schematically in For a 2DEG with Rashba and Dresselhaus SOIs, we find Nanostructured antidot lattices, consisting of periodically arranged holes that are etched in a 2DEG, form a strongly repulsive egg-carton-like periodic potential in a 2DEG3031323334353632xGax1−As/GaAs parabolically graded QW, which was fabricated successfully before3839V:We consider the 2DEG in a GaAs/Inm, and η iswhere ands see . The thiIn component, which behaves like an effective local electric field. This local electric field would not push the electron and the hole states to the left and right sides of the QW, but it can induce a considerably large ISOI hidden in symmetric QWs. The initial where n th miniband formed by the antidot lattice. We model the triangular antidot lattice potential V032414243a is the triangular antidot lattice constant (see Methods).The first and second subbands both form minibands due to the Brillouin zone folding caused by the antidot lattice. The band inversion could occur between the two adjacent minibands of the first subband k · p model to the following effective Hamiltonian within the basis To describe the four minibands (two spin-degenerate minibands) D = 0 BHZ Hamiltonian. Here A characterize the ISOI strength between neighboring minibands with opposite spin. This Hamiltonian obviously has a Z2 topological phase when M < 0, corresponding to band inversion.see , which ae ,  see , , and Ak · p model to calculate the subband structure with SOIs in a 40-nm-thick GaAs/InxGax1−As/GaAs parabolically graded QWs38k · p Hamiltonian by including the lowest 20 electron subbands and 54 highest hole subbands in the QW, to reproduce the energy dispersions of the first and second subbands calculated from the eight-band Kane model  in the first Brillouin zone shown in N spin-degenerate minibands being occupied, the Z2 invariant is given by mth occupied miniband at Γi. Our calculation gives To demonstrate that these minigaps are topologically nontrivial, we determine the parity of each miniband at the four time-reversal invariant momentax axis and y axis). As shown in Next, we demonstrate the emergence of topological edge states upon etching the QW into a Hall bar structure along two different directions , but the second minigap is larger (about 5 meV). By tuning the period and potential height of the antidot lattice, the nontrivial minigaps can be significantly enhanced see . For exa82Se3 and Bi2TexSe34950One way to detect the aforementioned edge states shown in  is the s8xGax1−As/GaAs QW, with antidot lattices created by well-developed etching technique. The key point is to create a ISOI in a symmetric quantum well, in contrast to conventional SOI in asymmetric QWs. This hidden ISOI in symmetric QWs induces a spin-dependent effective Lorentz force on the electrons, and generates the TI phases in such system. Interestingly, such ISOI exists in conventional semiconductors with a positive bandgap, i.e., normal band structures can generate quite large nontrivial gaps approaching 20 meV. This make it possible to observe the quantum spin Hall effect in liquid nitrigen temperature regime.Our proposal is based on a general analysis about the electron orbital motion in TIs. By using the Born-Oppenheimer approximation, we find that the fast motion will induce a spin-dependent gauge field on slow orbital motion. Based on this general analysis, we demonstrate theoretically the TI phase in a conventional 2DEG embedded in a symmetric parabolically graded GaAs/InSo far, all members of TI family are narrow bandgap systems containing heavy atoms. Our proposal breaks this constraint, and makes it possible to realize TI phase in conventional semiconductor 2DEG using the well-developed semiconductor fabrication techniques. The presence of the TI phase in parabolically graded QWs with antidot lattice can largely advance the application of this new quantum state in existing electronics and optoelectronics devices. The general designing principle proposed in this work, i.e., the gauge field acting on slow orbital motion induced by interband coupling, paves a new way for generating nontrivial topological phases, such as quantum spin Hall phase and even quantum anomalous Hall phases by doping magnetic ions, in conventional semiconductor 2DEGs, and suggests a promising approach to integrate it in well developed semiconductor electronic devices.z axis), effective spin-orbit coupling exists between subbands with opposite parities. This effective spin-orbit coupling comes from interband coupling and can be understand by reducing the 8 × 8 Kane Hamiltonian to a 2 × 2 effective Hamiltonian.For a symmetric quantum well grown along (001) direction  bands, with represents the interband coupling. Specifically, The eigenvalue problem can be expressed asz direction, e.g., a parabolically graded QW. By straightforward algebra, we have where η is given in Eq. σi denotes the real electron spin, and τi refers to the Pauli matrix describing the the subband index.Since we focus on the lowest conduction subbands, we have We consider a parabolically graded QW in the presence of an antidot lattice, which can be generally described by a potential k = 0, Γ point) is n = 2, 3, 4, 5, 6, 7) at the band edge are linear combinations of the six wave vector components For a triangular antidot lattice, the reciprocal lattice vectors in the hexagonal Brillouin zone are M at the Γ point, m* near the band edge, and A characterize the intersubband spin-orbit coupling. At the Γ pointThe lowest two subbands The accurate coupling strength can be estimated by numerical calculating based on the eight-band Kane model.Z2 topological transition from the normal phase to the topological insulator phase would occur when For BHZ model, a hen  see  changes Z2 invariants. For 2D systems, Fu and KaneZ2 invariant can be determined from the parity of the occupied band at the four time-reversal invariant momenta in the Brillouin zone. The Z2 invariant Topological insulators with dissipationless edge states and ordinary insulators are distinguished by different mth occupied energy band at the time-reversal invariant point Γi, which shares the same eigenvalue mth  occupied energy band at Γι are listed:where Z2 invariant ν = 1 at the 2mth  occupied band where the minigaps open, and the system enters the TI phase and the dissipationless edge states appear.From the above calculation, we can confirm that the How to cite this article: Shi, L. et al. Artificial Gauge Field and Topological Phase in a Conventional Two-dimensional Electron Gas with Antidot Lattices. Sci. Rep. 5, 15266; doi: 10.1038/srep15266 (2015).


The facilitated glucose transporter GLUT1 (SLC2A1) is an important mediator of glucose homeostasis in humans. Though it is found in most cell types to some extent, the level of GLUT1 expression across different cell types can vary dramatically. Prior studies in erythrocytes—which express particularly high levels of GLUT1—have suggested that GLUT1 is able to form tetrameric complexes with enhanced transport activity. Whether dynamic aggregation of GLUT1 also occurs in cell types with more modest expression of GLUT1, however, is unclear. To address this question, we developed a genetically encoded bioluminescent Förster resonance energy transfer (BRET) assay using the luminescent donor Nanoluciferase and fluorescent acceptor mCherry. By tethering these proteins to the N-terminus of GLUT1 and performing saturation BRET analysis, we were able to demonstrate the formation of multimeric complexes in live cells. Parallel use of flow cytometry and immunoblotting further enabled us to estimate the density of GLUT1 proteins required for spontaneous oligomerization. These data provide new insights into the physiological relevance of GLUT1 multimerization as well as a new variant of BRET assay that is useful for measuring the interactions among other cell membrane proteins in live cells. The efficiency of glucose uptake into cells is a major determinant of bioenergetic homeostasis. Members of the facilitated glucose transporter/solute carrier 2A (GLUT/SLC2A) family of proteins passively transport glucose across the plasma membrane, primarily enabling glucose uptake from systemic circulation234Several overlapping—and sometimes competing—mechanisms regulate the activity of GLUT1 in glucose transport. First among these is the differential expression of GLUT1 in distinct cell types, which can vary by orders of magnitude, for example in cancer versus normal cells68A second mechanism of GLUT1 activation involves the regulation of its subcellular distribution between internal vesicular compartments and the cell surface. Unlike the abundance of GLUT4 SLC2A4) at the membrane, which is more binary in nature and acutely triggered by extracellular signals such as insulin, the abundance of GLUT1 on the cell surface appears to be continuously titrated in response to the bioenergetic status of the cell, particularly with regards to the critical variable of cytoplasmic [AMP]/[ATP] ratioA4 at theA third mechanism that impinges on the activity of GLUT1 involves its ability to form higher-order multimeric complexes on the plasma membrane1113Renilla luciferase (Rluc) and a green fluorescent protein (GFP) derivative such as the yellow fluorescent protein (YFP)An efficient and simple method to quantify the number of molecules required for physical interaction to occur at the plasma membrane involves the use of bioluminescent Förster resonance energy transfer (BRET) between a luciferase donor and a fluorescent acceptor16In this study we describe a new version of the traditional BRET assay using a small 19 kDa) monomeric luciferase derived from deep-sea shrimp  as a donor and the well-characterized red fluorescent protein mCherry as an acceptor kDa mono1921E) between a given donor/acceptor pair, and the distance at which this efficiency is at 50%, defined as the Förster radius (R0)R) and the Förster radius.The quantitative principles governing BRET reactions are based upon equations developed by Theodor Förster for determining resonance energy transfer between two fluorophores (FRET)160), the refractive index of the assay medium (n), and the degree of overlap (J) between the donor emission spectrum and acceptor excitation spectrum.The Förster radius is a unique value for every donor/acceptor pair that can be calculated using four essential variables that are specific to a given BRET assay (equation 2). These variables include the orientation factor (κ) between the donor and acceptor, the quantum yield of the donor luciferase 20Previous BRET pairs using firefly or Among the various monomeric fluorescent acceptor proteins we considered, we chose to work with mCherry as an acceptor due to its large spectral separation from Nluc . Using mERET) for each fusion construct according to equation 3, which relates the ratio of donor emission to acceptor emission (410/610 nm) in the presence of either the donor alone (ID) or both the donor and acceptor (IDA).To empirically test the efficiency of the Nluc-mCherry BRET pair and validate its predicted Förster radius, we produced a series of mCherry-Nluc fusion proteins linked by a repeat peptide of known dimensionsA plot of these data as a function of the number of hexapeptide linker repeats clearly demonstrates that increasing the number of repeats leads to a decrease in BRET efficiency, as would be predicted by the Förster equation .D = 0.0033 +/− 0.0003), which allows us to measure changes in BRET ratio without dampening due to high background noise (The hexapeptide linker (GGSGGS) produces a tertiary fold of known diameternd noise . These dThe hexapeptide linker series we created produces data that fit well in the linear portion of the Förster equation (equation 1), allowing us to produce a standard curve by which the distance between Nluc and mCherry can be calculated using linkers of unknown dimensions . With th27We noticed that the BRET signal produced by our mCherry-GLUT1-Nluc fusion protein increased as a function of increasing plasmid transfection . This fiTo explain this unexpected result, we hypothesize that two modes of resonance energy transfer are occurring in cells transfected with mCherry-GLUT1-Nluc. The corrected BRET ratio produced at low levels of expression reflects “intramolecular BRET” between the N- and C-termini of GLUT1 molecules, which remains constant when mCherry-GLUT1-Nluc exists in a monomeric state. In contrast, the increased BRET signal seen with increasing expression levels of mCherry-GLUT1-Nluc can be accounted for by “intermolecular BRET” between molecules of a multimer in which light emitted from the Nluc donor on one mCherry-GLUT1-Nluc protein activates the fluorescence of mCherry on a juxtaposed fusion protein . These dGiven the saturation kinetics we observed for intermolecular BRET with the dual-labeled GLUT1 fusion protein, we hypothesized that it might be possible to estimate the number of GLUT1 molecules required to induce multimerization on the surface of 293FT cells. In this system, the number of mCherry molecules is equivalent to that of the GLUT1 proteins to which they are fused, indicating that accurate quantification of mCherry abundance relative to the BRET ratio would provide an estimate of the number of fusion proteins necessary to induce intermolecular BRET, which serves as a proxy for oligomerization. To quantify the average number of mCherry-GLUT1-Nluc proteins present in cells after transfection, we produced a standard curve using mCherry-coated beads whose fluorescence could be measured by flow cytometry . These b50 value of 8.40 × 105+/−1.58 × 105 molecules/cell, suggesting that approximately this number of GLUT1 proteins must be expressed in a 293FT cells in order for 50% of the molecules to be involved in multimeric complexes. Importantly, parallel experiments with a mCherry-2xlinker-Nluc fusion protein connected by two hexapeptide repeats failed to display saturation BRET kinetics, and instead produced a linear curve fit in which the BRET ratio remained the same regardless of the number of fusion protein expressed  and fit them to a hyperbolic saturation curve . These dxpressed . These dTo further confirm that intermolecular BRET could be used to investigate the multimerization kinetics of GLUT1, we individually fused either Nluc or mCherry to the N-terminus of GLUT1 . When excis transfection) or into two separate cell populations that were subsequently mixed at a 1:1 ratio (trans transfection). Using filtered luminescence on our plate reader, we then measured the donor (410/80 nm filter) and acceptor (610 nm long-pass filter) emission signals generated by addition of the Nluc substrate furimazine. In addition we used the standard monochromater function on the same plate reader to measure mCherry fluorescence after excitation with 587 nm light. As expected, we observed a dose-dependent increase in total Nluc emission and mCherry fluorescence as a function of the amount of DNA transfected into each well  that can be achieved is a function of distance between the donor and acceptor, we reasoned that saturation BRET assays using different combinations of Nluc and mCherry tagged GLUT1 could be used to test this model of GLUT1 oligomerizationPrior studies of GLUT1 in human erythrocytes have suggested that GLUT1 monomers interact in a specific orientation to form dimers, which then further organize into tetramers11BRETmax) and effective concentration at half BRETmax(EC50) could be determined in terms of plasmid ratios. In this context, BRETmax values are proportional to the distance between donor and acceptor, whereas EC50 values represent the relative affinity between donor and acceptor. These experiments clearly demonstrate that the closest association and highest relative affinity between Nluc and mCherry are achieved by transfecting cells with GLUT1 monomers bearing both fusion tags on N-termini  transposable element vector (pT2) to allow for stable expression in 293FT-HEK cells29GB1) with puromycin, we induced expression of mCherry-GLUT1 with a 2-fold serial dilution of doxycycline for 48 hours to determine the amount of drug required to saturate cells with Glut1. Interestingly, we found that increased expression of the mCherry-GLUT1 fusion protein by doxycycline also resulted in accumulation of both Nluc-GLUT1 and endogenous GLUT1  has become a method of choice for studying protein-protein interactions between membrane proteins due to its ability to be measured in live cells using a simple plate reader format. Previous iterations of the methodology using firefly or In this study, we describe a new variant of “nanoBRET” using Nluc and the red fluorescent protein mCherry. This specific BRET pair was chosen due to the large spectral separation between Nluc and mCherry emission maxima, which generates an exceedingly low background BRET ratio using the filter sets described above. This characteristic is important for generating reproducible BRET data across a large range of acceptor concentrations, particularly at concentrations far below saturation. Our empirical values for this BRET pair fit remarkably well with theoretical predictions made by the Förster equation, allowing us to calculate parameters for homotypic interactions between GLUT1 molecules with high confidence.et al. which suggests that GLUT1 monomers initially associate as N-to-N terminal dimers, and then form tetramers stabilized by homotypic interactions between juxtaposed C-termini of the four monomers  in place of the V5 epitope tag. This allowed us to use the GATEWAY cloning system for subsequent recombination of the mouse Glut1 open reading frame  from the pCR8-TOPO entry vector into each of these vectors to create individual GLUT1 fusion proteins with Nluc or mCherry at either terminus. For the dual fusion vector (pcDNA6.2-mCherry-Glut1-Nluc), the open reading frame for mCherry was inserted in frame upstream of Glut1-Nluc in the pcDNA6.2-Glut11-Nluc vector using standard restriction cloning methods.Vectors containing the open reading frames for Nanoluciferase  and mCherry (pmCherry) were obtained commercially from gBlock double-stranded DNA sequence (Integrated DNA Technologies) by Gibson cloning (New England Biolabs). This new insert contained one copy of the hexapeptide coding sequence (GGCGGCAGCGGCGGATCC) connecting the open reading frames of mCherry and NanoLuc, and was located upstream of BamH1 and Xba1 sites immediately proximal to the 5′ end of NanoLuc. Subsequent hexapeptide repeats were inserted by digesting the vectors with BamH1 and Xba1 and inserting in an annealed, double-stranded oligonucleotide encoding a single hexapeptide repeat flanked by overhangs homologous to the BamH1 and Xba1 restriction sites. Insertion of this oligonucleotide extended the repeat by a single unit in frame with the mCherry and Nluc proteins and re-introduced new 3′ BamH1 and Xba1 sites, while at the same time destroying the 5′ BamH1 site. Repeated iterations of this protocol allowed for creation of a series of linker vectors in which mCherry and Nluc were connected by a linker of 1–10 repeats of the hexapeptide.The fusion vectors in which mCherry and Nluc were linked together by a defined series of hexapeptide (GGSGGS) repeats were created by removing GLUT1 from pcDNA6.2-mCherry-Glut1-Nluc via restriction digest, and replacing it with a Addgene, #26557)Clontech) into pT2-EF1α to create a constitutively expressing stable vector for tetracycline/doxycycline inducible expression of mCherry-GLUT1. Partial maps for these three vectors are shown in Addgene, #34879), which encodes the SB100X transposaseThe stable transposable element vectors based on the Sleeping Beauty system were created by subcloning the expression cassettes for mCherry-GLUT1 and Nluc-GLUT1 into the pT2-HB vector (obtained from Dr. Perry Hackett via Invitrogen) used in this study were cultured in Dulbecco’s Modified Eagle Medium , which was supplemented with 10% fetal bovine serum . Cells were split three times per week to maintain log-phase growth, and seeded at specific densities after counting with a standard hemocytometer. Transient transfections into 293FT-HEK cells were performed using LipoD293 cationic lipid reagent (SignaGen Laboratories) at a ratio of 3 μL of reagent per 1 μg of plasmid DNA. Transfections into cells plated to 96-well plates (see below) were performed in a final volume of 100 μL per well, whereas transfections into cells in 6-well plates were done in a final volume of 2 mL. In both cases, media was subsequently changed 18–24 hours after transfection to remove excess cationic lipid from the media. Stable insertion of transposable elements was selected for by culture of cells in media containing 2.0 μg/mL of puromycin (Sigma-Aldrich).The 293FT-HEK cells (4 per well (Greiner Biosciences). Cells were transfected in triplicate with expression plasmids (50 ng per well) at 18–24 hours post-seeding. The following day (~24 hours post-transfection), media was removed and replaced with fresh complete media. At 48 hours post-transfection media was again removed and replaced with serum and phenol red-free DMEM media, and cells were allowed to equilibrate for one hour under normal culture conditions. The cells were subsequently treated with 5 nm furimazine (Promega) and immediately measured for emission of light at 410 and 610 nanometers using a Synergy Hybrid H1 plate reader (BioTek) equipped with a 410/80 and 610-long pass luminescence filter set. The corrected BRET ratio was determined by subtracting the basal 610/410 filtered luminescence value of GLUT1-Nluc alone (ID) from the 610/410 ratio of cells also expressing mCherry-fusion acceptors (IDA) at various levels.BRET assays were uniformly performed in white-walled, microclear-bottom 96-well plates into which 293FT cells were seeded at a density of 1.0 × 106 cells were transfected in 6-well plates with volumes scaled according to the surface area ratio of 96-well to 6-well plates (1:100). At 24 hours after transfection, the cells were reseeded in parallel to a new 6-well plate and in triplicate to a 96-well assay plate at densities of 1.0 × 106 and 2.0 × 104 cells per well, respectively. The BRET assays were performed 18–24 hours after replating as indicated above.For BRET assays performed in parallel with flow cytometry to allow for calculation of mCherry expression density, 1.0 × 104 cells/well and transfected 24 hours later with 1.0 μg/well of plasmid DNA for each GLUT1 fusion protein. After 48 hours, the cells were detached and replated to 24-well dishes at a density of 5.0 × 104 cells/well and allowed to adhere and equilibrate to culture conditions for 18–24 hours. Glucose uptake was measured using the radiolabeled glucose analog 2-deoxyglucose (2DG), which was added with 14C-mannitol as a control for cell membrane integrity. Briefly, the media was replaced with 0.3 mL of glucose-free HEPES buffer [pH 7.4]  supplemented with 1.0 mM (0.3 μCi/mL) 2-DG  and 1.0 mM (0.02 μCi/mL) mannitol (1-14C). After a 10 minute incubation, cells were washed twice with cold glucose-free HEPES. The cells were digested in 0.25 mL 0.3 M NaOH prior to measuring the 3H-2DG uptake and 14C-mannitol background signal. Quadruplicate 3H-2DG uptake values corrected for 14C-mannitol binding were averaged and tested for significance using student’s T-test.HEK-293FT cells were plated to 6-well dishes at a density of 5.0 × 10Invitrogen/Life Technologies). Cells were subsequently filtered to achieve a single cell suspension, and immediately assayed on a FACScalibur flow cytometer (Becton Dickinson). Each cell population was gated according to forward and side scatter profile to identify intact cells, which were then measured on the FL2 channel to quantify mCherry expression. No fewer than 2.0 × 104 cells were captured for each sample, and each condition was measured in triplicate to obtain a mean fluorescent intensity (MFI) for the population. Mean fluorescent intensities were translated into molecular equivalents of soluble fluorophore (MESF) using calibrated mCherry beads (Clontech) with defined MESF values. The beads were run for each assay replicate using the same laser settings that were used to quantify mCherry expression in transfected cells.Transfected 293FT cells were replated to 6-well dishes and allowed to adhere and equilibrate to culture conditions for 18–24 hours. Cells were rinsed with PBS and detached from the plate in 1 mL of cold versene Transfected cells in 6-well format were rinsed once with PBS and directly scraped into 0.1 mL lysis buffer  supplemented with fresh protease inhibitor cocktail (Sigma-Aldrich). The lysate was homogenized by sonicating for 5 seconds while incubating on ice, and then cleared by centrifugation at 10,000 × g for 5 minutes in a chilled centrifuge at 4 °C. Soluble protein was removed to a fresh tube on ice, quantified by Bradford assay and denatured for 10 minutes at 95 °C in a dry bath. The samples were then deglycosylated with recombinant PNGaseF (prepared from GraphPad Software) for graphing and statistical analysis. Data were best fit to standard hyberbolic or sigmoidal curves with correlation coefficients indicated in each legend. EC50 values with 95% confidence intervals were obtained from triplicate or quadruplicate experiments as indicated and, when relevant, analyzed for significance using student’s T-test.Data for each experiment were exported into the Prism software package .


The RKB rhizosphere diazotroph assemblage was very diverse and apparently consists mainly of the genera Rhizobium, followed by unknown diazotrophic microorganisms. Deduced amino acid sequence analysis revealed the presence of diverse nifH sequences, affiliated with a wide range of taxa, encompassing members of the Proteobacteria, Actinobacteria and Firmicutes. Members of cyanobacteria, methanotrophs and archaea were also detected. To the best of our knowledge, this is the first major metagenomic effort that revealed the presence of diverse nitrogen-fixing microbial assemblages in indigenous RKB rhizospheric soil which can further be explored for improved crop yield/productivity.Red kidney beans (RKBs) are one of the major components in the human diet of Western Indian Himalaya (WIH). Their cultivation in these habitats is strongly influenced by various biotic and abiotic stresses and therefore, there must be a selection of RKB associated microorganisms that are adapted to these harsh conditions. Seven cold adaptive diazotrophs from the same rhizosphere were isolated in our previous study to reveal the low-temperature associated proteins and mechanisms. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are still unknown. In this study, RKB rhizospheric soil from two different agro-ecosystems of WIH namely S1  and S2  were explored for the assessment of nitrogenase reductase gene (The online version of this article (doi:10.1007/s13205-014-0238-5) contains supplementary material, which is available to authorized users. Recently, seven cold adapted bacterial diazotrophs from the RKB rhizosphere of WIH were isolated and proteome of the psychrophilic nitrogen-fixing Pseudomonas migulae strain S10724 for low-temperature diazotrophy was documented , complex carbohydrates (50–60 %) and a better source for vitamins, minerals and poly unsaturated free fatty acids  has become one of the largest non-ribosomal gene datasets on uncultivated microorganisms  using sterile spatula in sterile polythene bags and transported to laboratory under sterile and cold conditions. Each soil sample was collected in triplicates which were later mixed to make a single composed sample per site. Samples for chemical analysis were stored at 4 °C, and samples for clone library analysis were stored at −20 °C till further use.The sampling sites were located on the upper reaches of Kumaun Himalaya Fig. . RhizospSoil samples (1 g) were analyzed chemically at Accurate Analytical Laboratory, Pune  for pH, total carbon (TC), nitrogen (TN), phosphorus (TP) and potassium (K) contents.nifH library construction; remaining DNA was stored in TE buffer  at −80 °C till further use.Total soil DNA was extracted from each 0.5 g (fresh weight) soil sample by using the Powersoil™ DNA isolation kit , according to the manufacturer’s instructions. After extraction, DNA samples were quantified spectrophotometrically at 260 nm and used immediately for nifH genes from collected soil samples was quantified using iCycleriQ™ Multicolor  real-time polymerase chain reaction (qPCR) machine as described previously . PCR reactions was carried out in a volume of 25 μl containing 1X iQTM SYBR Green Supermix (Bio-Rad), 100 pmol of each nifH primer PolF (5′ TG GAY CCS AAR GCB GAC TC 3′), PolR (5′ ATS GCC ATC ATY TCR CCG GA 3′) and 50 ng of template DNA. Cycling conditions included 5 min of the denaturation step at 95 °C, followed by 30 cycles of 30 s at 95 °C, 30 s at 50 °C and 30 s at 72 °C  by using the ‘TOPO TA cloning®’ kit (Invitrogen) for sequencing with ‘One Shot TOP10 Electro-Competent cells®’, according to the manufacturer’s instructions (Invitrogen) on the same day. Randomly selected colonies were picked and subjected to colony PCR in 25 μl of PCR mixture as described earlier  version 1.77  technique (Table SM1). Bacterial gene abundances act as the indicators of various processes occurring in the respected ecosystem and therefore, real-time PCR becomes an effective and sensitive presumptive screening tool for estimating microbial abundance  and Munsyari, located at the bottom of “Milam” glacier, respectively. Physicochemical analysis of S1 and S2 revealed the significant difference between the rhizospheric soils of both the agro-ecosystems. Large difference among the total nitrogen (N) and total organic carbon (TOC) content of both the soils were recorded. However, total phosphorus (P) and potassium (K) content differ less significantly . Upon excluding the uncultured/environmental sample sequences from the queries, most of them showed sequence homology with their nearest cultivable representatives  and Firmicutes (Bacillus). In addition to this, an archaeal MethanobrevibacternifH sequence was also encountered in SN1. Cyanobacterial nifH sequences belonging to the genera Lyngbya, Synechococcus and Plectonema were exclusively observed in SN2 library.A total of 196 positive clones were obtained from two clone libraries SN1 (87 sequences) and SN2 (109 sequences); constructed using the soils S1 and S2, respectively. A good fraction of the clone sequences belonged to unculturable NDetermination of operational taxonomic unit (OTU) is one of the preferred methods that are currently available for comparing diversity from different clone libraries. Diversity is a function of the number of OTUs present (OTU richness) and the evenness with which clones are distributed among these OTUs (OTU evenness) Hurlbert . Based onifH amino acid sequences from SN1 and SN2 were compared with sequences in the databases and their phylogenetic relationships were investigated. Both the libraries SN1 and SN2 showed similar type of nifH distribution pattern. All the sequences were clustered into four major groups designated as groups I, II, III and IV  nifH sequences into two different groups: group I and IV. Similarly, in SN1 genus Dechloromonas was also clustered into two groups I and IV. Moreover, cyanobacteria nifH sequences were found to be distributed in two major groups II and III of SN2. Additionally, the sequences of Bacillus (Firmicutes) were found clustering with sequences of Methylocaldum (Gammaproteobacteria) as well as Rhizobium (Alphaproteobacteria) in Group I of SN1. Genus Arthrobacter (Actinobacteria) was found to cluster with Methanobrevibacter (archea) in SN1 and cyanobacteria group in SN2. At the same time, though belong to same phylogenetic group (16SrRNA based), nifH sequences of Rhizobium and Bradyrhizobium were separated apart in both the libraries. These anomalies between nifH phylogeny and ribosomal RNA phylogeny could be the result of lateral gene transfer among microorganisms. It is not clear whether these nitrogenase genes from one cluster can be distinguished from that of other. But, this type of distribution of single genera in multiple clusters may be justified in terms of the presence of pseudo-nifH and/or nifH like gene(s) that are considered to function in some process other than nitrogen fixation Below is the link to the electronic supplementary material.